Anti-Extended Type I Glycosphingolipid Antibody, Derivatives Thereof And Use

ABSTRACT

Human antibodies and antigen-binding portions of those antibodies that specifically bind extended Type I chain glycosphingolipids are provided.

FIELD OF THE INVENTION

The present invention relates to anti-extended Type I glycosphingolipidantibodies and their use in the amelioration, treatment or prevention ofdiseases or disorders in mammals, including humans, resulting fromimproper activity or metabolism of same; resulting, causing orassociated with; or the presence thereof, for example, in a cancer, suchas colorectal cancer, or other pathology. An antibody of interest can beused for therapeutic purposes or diagnostic purposes. Thus,prophylactic, immunotherapeutic and diagnostic compositions comprisingthe antibodies and derivatives thereof of interest and their use inmethods for preventing or treating, or diagnosing diseases in mammals,including humans, caused by inappropriate metabolism and/or expressionof extended Type I glycosphingolipid in and on cells, such as certainmalignant cells, also are disclosed.

BACKGROUND

Extended Type I glycosphingolipid is a cell surface molecule that can beassociated with, for example, certain malignant states.

Aberrant glycosylation has been observed to be a common feature of manycancer types, Hakomori, PNAS 99:10231-10233, 2002. Some of thecarbohydrate antigens used for the diagnosis of human cancers carrypolylactosamine structures. Polylactosamines are usually classified intotwo categories according to the unit structure. A polylactosamine havingthe Galβ1→3GlcNAc structure is called a Type I chain, and that havingthe Galβ1→4GlcNAc structure is referred to as a Type II chain. The mostcommon tumor-associated antigens found in some human cancers have thelacto series Type II chain structure, which usually is sialylated and/orfucosylated. Type I chain antigens are abundant in normal cells andtissues, and occasionally are associated with cancer, Stroud et al., JBC266: 8439-8446, 1991. For example, 2→3 sialylated Le^(a) antigen (the CA19-9 antigen defined by the N19-9 antibody) is a cancer-associated TypeI chain antigen. However, cancer diagnostic methods based on thedetection of those Type I antigens have been hampered by high falsepositive and/or high false negative incidences, see, for example U.S.Pat. Nos. 6,083,929 and 6,294,523.

Two mouse monoclonal antibodies, NCC-ST421 and IMH2, were raised againstextended Type I chain antigens. NCC-ST421 is specific for Le^(a)-Le^(a).The NCC-ST421 antibody strongly induced antibody dependant cellcytotoxicity (ADCC) using human peripheral blood leukocytes as effectorsagainst a variety of human tumor cells, and induced complement dependentcytotoxicity (CDC) with a human complement source, Watanabe et al.,Cancer Res. 51:2199-2204, 1991. The Le^(a)-Le^(a) antigen was found tobe highly expressed in the human colon carcinoma cell line, Colo205.

IMH2 was also established against extended Type I chains. IMH2 bound toLe^(b)-Le^(a), Le^(y)-Le^(x), Le^(b) and Le^(y) based on ¹H-NMR, FAB-MSand enzymatic degradation studies, Stroud et al., Eur. J. Biochem.203:577-586, 1992. IMH2 showed strong lymphocyte-activated, as well as,complement-dependent killing of Colo205 cells in vitro, and inhibitedColo205 growth in vivo.

IMH2 reacted with carcinoma tissues derived from colon, pancreas, liverand endometrium. However, normal colon showed no reactivity with IMH2.Normal liver and pancreas showed weak or highly restricted reactivity innormal hepatocytes and islets of Langerhans cells. Immunochemicalstaining intensity was much stronger in endometrial carcinomas than innormal endometrium, Ito et al., Cancer Res. 52:3739-3745, 1992.

Both NCC-ST421 and IMH2 exhibit inhibition of tumor growth in nude miceafter inoculation of human tumor cells expressing the extended Type Ichain antigen, but no inhibition of growth occurred in tumor cells thatdid not express extended Type I chain antigen.

Because of the abundance of Type I structures on normal cells, the useof Type I antibodies for diagnostic and/or therapeutic purposesheretofore was not possible.

Conventional cancer treatments, such as chemotherapy and radiotherapy,have shown some advantages in various cancer patients. Despite thebenefits of antitumor activity in conventional therapies, however,treatment-induced toxicity to normal tissues can substantially reducethe quality of life in cancer patients. Dose intensification for betterantitumor activity is also limited. Monoclonal antibodies enable thepromise of targeted cytotoxicity, focusing on tumor tissues, but notnormal tissues.

Monoclonal antibodies (mAbs) can be developed with high specificity forantigens expressed on tumor cells and can elicit desired antitumoractivities. The promise of mAbs was furthered by the development of micethat produce fully human mAbs. One such tool is the KM mouse, U.S. Pat.No. 7,041,870 and Tomizuka et al., Nat. Genet. 16:133-143, 1997. In theKM mouse, the mouse genes encoding immunoglobulins were inactivated andreplaced with human antibody genes. Thus, the KM mouse expresses fullyhuman antibodies.

Several fully human antibodies have been successfully developed usingthe KM mouse.

For example, Motoki et al. developed a human IgG (KMTR2) which directedantibody dependent oligomerization of TRAIL-R2 and initiated efficientapoptotic signaling and tumor regression independent of host effectorfunction (Clin. Cancer Res. 11(8):3126-3135, 2005; and see U.S. Pat. No.7,115,717 and Imakire et al., Int. J. Cancer 108:564-570, 2004). HD8, afully human monoclonal antibody specific for human leukocyte antigen DR(HLA-DR), exerted antibody-dependent cellular cytotoxicity (ADCC) aswell as complement-dependent cytotoxicity (CDC) in vitro, and extendedthe life span of immunocompromised mice inoculated with non-Hodgkinlymphoma cell lines, Tawara et al., Cancer Sci. 98 (6) 92′-928, 2007.

Additionally, two human IgMs raised in KM mice and directed tocarbohydrate antigens were reported. HMMC-1 specifically recognizes anovel O-glycan structure, reacts positively with Mullerian duct-relatedcarcinomas, and exhibits complement dependent cytotoxicity on a humanuterine endometrial cancer cell line, SNG-S, Nozawa et al., Clin Cancer.Res. 10:7071-7078, 2004. Another human monoclonal IgM, HMOCC-1,recognizing a glycoprotein located on the cell membrane, reacted withovarian cancer (Suzuki et al., Gynecol. Oncol. 95:290-298, 2004). Sincethose two antibodies are IgMs, the application of those antibodies incancer therapy should be limited by molecule size and restrictions inproduction.

SUMMARY

The present invention provides novel human antibodies, and fragments andderivatives thereof, that specifically bind to extended Type Iglycosphingolipid.

The invention includes the amino acid sequences of the variable heavyand light chain of the antibodies and their corresponding nucleic acidsequences.

Another embodiment of the invention includes the complementaritydetermining regions (CDR) sequences of the antibodies of interest toobtain binding molecules that comprise one or more CDR regions, orCDR-derived regions, that retain extended Type Iglycosphingolipid-binding capacity of the parent molecule from which theCDR's were obtained.

Another embodiment of the present invention includes the cell lines andvectors harboring the antibody sequences of the present invention.

Another embodiment of the present invention relates to the use of theantibodies for the preparation of a medicament or composition for thetreatment of diseases and disorders associated with extended Type Iglycosphingolipid function, metabolism and expression.

Another embodiment of the present invention relates to the use of theantibodies in the diagnosis of disorders associated with atypical orabnormal extended Type I glycosphingolipid biology and expression.

Those and other goals were met in the development of human monoclonalantibodies against extended Type I chain carbohydrate antigens. Forexample, mAb GNX-8 is a human IgG1 derived from a KM mouse. GNX-8exhibits CDC and ADCC activity on several human colorectal cancer celllines and inhibits Colo205 and DLD-1 tumor growth in vivo. GNX-8 reactswith primary and metastatic colorectal cancers, breast cancers, pancreascancers as well as lung cancers, but not with normal human tissues andblood cells.

Additional features and advantages are described herein, and will beapparent from, the following Detailed Description.

DETAILED DESCRIPTION

The invention is not limited to the particular methodology, protocols,polypeptides, polynucleotides, cell lines, vectors, or reagentsdescribed herein because variations can occur or can be used withoutdeparting from the spirit and scope of the invention. Further, theterminology used herein is for the purpose of exemplifying particularembodiments only and is not intended to limit the scope of the presentinvention. Unless defined otherwise, all technical and scientific termsand any acronyms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art in the field of theinvention. Any method and material similar or equivalent to thosedescribed herein can be used in the practice of the present inventionand only exemplary methods, devices, and materials are described herein.

All patents and publications mentioned herein are incorporated herein inentirety by reference for the purpose of describing and disclosing theproteins, enzymes, vectors, host cells and methodologies reportedtherein that might be used with and in the present invention. However,nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosures by virtue of prior invention.

An “extended Type I glycosphingolipid disease” is a malady, disorder,disease, pathology, condition, abnormality and so on, which ischaracterized by, associated with or caused by abnormal metabolism,overexpression or increased levels of extended Type I glycosphingolipid,for example, at the cell surface.

The phrase “substantially identical” with respect to an antibodypolypeptide sequence may be construed as an antibody chain exhibiting atleast 70%, 80%, 90%, 95% or more sequence identity to a referencepolypeptide sequence. The term with respect to a nucleic acid sequencemay be construed as a sequence of nucleotides exhibiting at least about85%, 90%, 95%, 97% or more sequence identity to a reference nucleic acidsequence.

The terms, “identity” or “homology” may mean the percentage ofnucleotide bases or amino acid residues in the candidate sequence thatis identical with the residues of a corresponding sequence to which thecandidate is compared, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent identity for theentire sequence, and not considering any conservative substitutions aspart of the sequence identity. Neither N-terminal nor C-terminalextensions nor insertions shall be construed as reducing identity orhomology. Methods and computer programs for the alignment of sequencesare available and are well known in the art. Sequence identity may bemeasured using sequence analysis software.

The phrases and terms, “functional fragment, variant, derivative oranalog” and the like, as well as forms thereof, of an antibody, nucleicacid or antigen is a compound or molecule having qualitative biologicalactivity in common with a full length antibody or antigen of interest.For example, a functional fragment or analog of an anti-extended Type Iglycosphingolipid antibody is one which can bind to an extended Type Iglycosphingolipid molecule, or is an agonistic or antagonistic antibodywhich binds to extended Type I glycosphingolipid. An example is anscF_(V) molecule. As to extended Type I glycosphingolipid, a variant orderivative thereof is a molecule that is not identical to a naturallyoccurring extended Type I glycosphingolipid and yet can be used for apurpose of the instant invention, such as, while not identical to a wildtype extended Type I glycosphingolipid nevertheless can be used, forexample, as immunogen to raise antibodies that selectively bind to wildtype extended Type I glycosphingolipid.

“Substitutional” variants are those that have at least one amino acidresidue in a native sequence removed and replaced with a different aminoacid inserted in place at the same position. The substitutions may besingle, where only one amino acid in the molecule is substituted, or maybe multiple, where two or more amino acids are substituted in the samemolecule. The plural substitutions may be at consecutive sites. Also,one amino acid can be replaced with plural residues, in which case sucha variant comprises both a substitution and an insertion.

“Insertional” variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative sequence. Immediately adjacent to an amino acid means connectedto either the α-carboxyl or α-amino functional group of the amino acid.

“Deletional” variants are those with one or more amino acids in thenative amino acid sequence removed. Ordinarily, deletional variants willhave one or two amino acids deleted in a particular region of themolecule.

The terms, substitution, insertion and deletion variants also applyanalogously to nucleic acids.

The adaptive immune response has two major arms: the cellular immuneresponse of T lymphocytes and the humoral immune response of antibodysecreting B lymphocytes. B cell epitopes can be linear, contiguous aminoacids, or can be conformational (Protein Science (2005) 14, 246). Incontrast, T cell epitopes are short linear peptides that are cleavedfrom antigenic proteins that are presented in the context of majorhistocompatibility complex (MHC) proteins, or, in case of humans, humanleukocyte antigen (HLA) class I or class II molecules. Epitopepresentation depends on both MHC-peptide binding and T cell receptor(TCR) interactions. MHC proteins are highly polymorphic, and each bindsto a limited set of peptides. Thus, the particular combination of MHCalleles present in a host limits the range of potential epitopesrecognized during an infection.

Two fundamental types of T cells are distinguished by expression of CD8and CD4 proteins, which dictate whether a T cell will recognize epitopespresented by class I or class II molecules, respectively. CD4⁺ Tepitopes are processed after encapsulation by antigen presenting cellsin membrane bound vesicles, where the antigen is degraded by proteasesinto peptide fragments that bind to MHC class II proteins. In contrast,CD8⁺ T cells generally recognize viral or self antigens expressed fromwithin a cell, proteins that are cleaved into short peptides in thecytosol by the immunoproteasome. After cleavage, peptides aretranslocated by the transporter associated with antigen processing (TAP)into the endoplasmic reticulum for loading onto HLA I antigens. CD4⁺ T(helper) cell epitopes are critical in driving T cell-dependent immuneresponses to protein antigens.

The term “antibody” is used in the broadest sense, and includesmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), antibody fragments or synthetic polypeptides carrying oneor more CDR or CDR-derived sequences so long as the polypeptides exhibitthe desired biological activity. Antibodies (Abs) and immunoglobulins(Igs) are glycoproteins having the same structural characteristics.Generally, antibodies are considered Igs with a defined or recognizedspecificity. Thus, while antibodies exhibit binding specificity to aspecific target, immunoglobulins include both antibodies and otherantibody-like molecules which lack target specificity.

The antibodies of the invention can be of any class (e.g., IgG, IgE,IgM, IgD, IgA and so on), or subclass (e.g., IgG₁, IgG₂, IgG_(2a), IgG₃,IgG₄, IgA₁, IgA₂ and so on) (“type” and “class,” and “subtype” and““subclass,” are used interchangeably herein). Native or wildtype, thatis, obtained from a non-artificially manipulated member of a population,antibodies and immunoglobulins, and monomers of polymeric antibodies,such as IgA and IgM, are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atthe other end.

By “non-artificially manipulated” is meant not treated by non-naturalmeans, such as immunization or transformation, to contain or to expressa foreign antigen binding molecule. Wildtype can refer to the mostprevalent allele or species found in a population or to the antibodyobtained from a non-artificially manipulated animal, as well as tonaturally occurring alleles or polymorphisms which arise naturally andcan be sustained in a population, or a variant or derivative arisingthrough natural means, such as a malignancy, as compared to thatobtained by a form of manipulation, such as mutagenesis, use ofrecombinant methods and so on to change an amino acid of theantigen-binding molecule. The use of the term is readily inferred andunderstood by the artisan in the context of the sentence, paragraph,concept, thought, idea and so on in which the term is found, used and soon.

As used herein, “anti-extended Type I glycosphingolipid antibody” meansan antibody or derived polypeptide which binds specifically to humanextended Type I glycosphingolipid.

The term “variable” in the context of a variable domain of antibodies,refers to certain portions of a pertinent molecule which differextensively in sequence between and among antibodies and can be integralin the specific recognition and binding of a particular antibody to aparticular target. However, the variability is not evenly distributedthrough the variable domains of antibodies.

The variability can be concentrated in three segments calledcomplementarity determining regions (CDRs; i.e., CDR1, CDR2 and CDR3)also known as hypervariable regions, both in the light chain and theheavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework (FR) regions or sequences. Thevariable domains of native heavy and light chains each comprise four FRregions, largely adopting a β sheet configuration, connected by threeCDRs, which form loops connecting, and in some cases forming part of,the β sheet structure. The CDRs in each chain are held together, oftenin proximity, by the FR regions and, with the CDRs from the other chain,contribute to the formation of the target (epitope or determinant)binding site of antibodies (see Kabat et al. Sequences of Proteins ofImmunological Interest, National Institute of Health, Bethesda, Md.(1987)). One CDR, such as, CDR3 of the heavy chain, can alone carry theability to bind specifically to the cognate epitope.

As used herein, numbering of immunoglobulin amino acid residues is doneaccording to the immunoglobulin amino acid residue numbering system ofKabat et al., unless otherwise indicated.

The term “antibody fragment” refers to a portion of an intact or a fulllength chain of an antibody, generally the target binding or variableregion. Examples of antibody fragments include, but are not limited to,F_(ab), F_(ab′), F_((ab′)2), and F_(v) fragments. A “functionalfragment” or “analog of an anti-extended Type I glycosphingolipidantibody” is one which can bind a cognate antigen. As used herein,functional fragment generally is synonymous with, “antibody fragment,”and with respect to antibodies, can refer to fragments, such as F_(v),F_(ab), F_((ab′)2) and so on which can bind a cognate antigen.

An “F_(v)” fragment consists of a dimer of one heavy and one light chainvariable domain in a non-covalent association (V_(H)-V_(L) dimer). Thatconfiguration of the three CDR's of each variable domain interact todefine a target binding site of the V_(H)-V_(L) dimer as in an intactantibody. Collectively, the six CDRs confer target binding specificityon the intact antibody. However, even a single variable domain (or halfof an F_(v) comprising only three CDRs specific for a target) can havethe ability to recognize and to bind target.

“Single-chain F_(v)” “sF_(v)” or “scAb” antibody fragments comprise theV_(H) and V_(L) domains of an antibody, wherein the domains are presentin a single polypeptide chain. Generally, the F_(v) polypeptide furthercomprises a polypeptide linker, often a flexible molecule, such as, anoligopeptides, which may be obtained from a naturally occurringmolecule, derived from a naturally occurring molecule, is an artificialsequence, such as polyglycine, and so on, between the V_(H), and V_(L)

The term “diabodies” refers to antibody fragment constructs with twoantigen binding sites, which fragments can comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain. By using a linker that is tooshort to allow pairing between the two variable domains on the samechain, the diabody domains are forced to pair with the binding domainsof another chain to create an antigen binding site.

The F_(ab) fragment contains the variable and constant domains of thelight chain and the variable and first constant domain (C_(H1)) of theheavy chain. F_(ab′) fragments differ from F_(ab) fragments by theaddition of a few residues at the carboxyl terminus of the C_(H1) domainto include one or more cysteines from the antibody hinge region. F_(ab′)fragments can be produced by cleavage of the disulfide bond at the hingecysteines of the F_((ab′)2) pepsin digestion product. Additionalenzymatic and chemical treatments of antibodies can yield otherfunctional fragments of interest.

The term “monoclonal antibody” (mAb or MAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts.

Monoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass (type or subtype), with the remainder of the chain(s) identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as the chimeric antibodiesexhibit the desired biological activity of binding to extended Type Iglycosphingolipid or impacting extended Type I glycosphingolipidactivity or metabolism (U.S. Pat. No. 4,816,567; and Morrison et al.,Proc Natl Acad Sci USA 81:6851 (1984)). Thus, CDR's from one class ofantibody can be grafted into the FR of an antibody of different class orsubclass.

Monoclonal antibodies are specific, being directed against a singletarget site, epitope or determinant. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes)of an antigen, each monoclonal antibody is directed against a singledeterminant on the target. In addition to their specificity, monoclonalantibodies are advantageous being synthesized by a host cell,uncontaminated by other immunoglobulins, and provide for cloning therelevant gene and mRNA encoding the antibody chains thereof. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies for use withthe present invention may be isolated from phage antibody librariesusing well known techniques or can be purified from a polyclonal prep.The parent monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method described byKohler et al., Nature 256:495 (1975), or may be made by recombinantmethods well known in the art.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such asF_(v), F_(ab), F_(ab′), F_((ab′)2) or other target-binding subsequencesof antibodies) which contain sequences derived from non-humanimmunoglobulin, as compared to a human antibody. In general, thehumanized antibody will comprise substantially all of one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulintemplate sequence.

The humanized antibody may also comprise at least a portion of animmunoglobulin constant region (F_(c)), typically that of the humanimmunoglobulin template chosen. In general, the goal is to have anantibody molecule of certain specificity that is minimally immunogenicin a human. Thus, it is possible that one or more amino acids in one ormore CDR's also can be changed to one that is less immunogenic to ahuman host, without substantially minimizing the specific bindingfunction of the one or more CDR's to extended Type I glycosphingolipid.

Alternatively, the FR can be non-human but those amino acids mostimmunogenic are replaced with ones less immunogenic. Nevertheless, CDRgrafting, as discussed above, is not the only way to obtain a humanizedantibody. For example, modifying just the CDR regions may not besufficient to optimize an antibody as it is not uncommon for frameworkresidues to have a role in determining the overall three-dimensionalstructure of the CDR loops and the overall affinity of the antibody forthe ligand.

Hence, any means can be practiced to reduce antibody immunogenicity sothat the non-human parent antibody molecule is modified to be one thatis less immunogenic to a human, and global sequence identity with ahuman antibody is not always a necessity. So, humanization also can beachieved, for example, by the mere substitution of just a few residues,particularly those which are exposed on the antibody molecule surfaceand not buried within the molecule, and hence, not readily accessible tothe host immune system. Such a method is taught herein with respect tosubstituting, for example, charged or certain other residues on theantibody molecule, the goal being to reduce or dampen the immunogenicityof the resultant molecule without compromising the specificity of theantibody for the cognate epitope or determinant. See, for example,Studnicka et al., Prot Eng 7(6)805-814, 1994; Mol Imm 44:1986-1988,2007; Sims et al., J Immunol 151:2296 (1993); Chothia et al., J Mol Biol196:901 (1987); Carter et al., Proc Natl Acad Sci USA 89:4285 (1992);Presta et al., J Immunol 151:2623 (1993), WO 2006/042333 and U.S. Pat.No. 5,869,619.

Strategies and methods for resurfacing antibodies, and other methods forreducing immunogenicity of antibodies within a different host, aredisclosed, for example, in U.S. Pat. No. 5,639,641. Briefly, in apreferred method, (1) position alignments of a pool of antibody heavyand light chain variable regions are generated to yield heavy and lightchain variable region framework surface exposed positions, wherein thealignment positions for all variable regions are at least about 98%identical; (2) a set of heavy and light chain variable region frameworksurface exposed amino acid residues is defined for a non-human, such as,a rodent antibody (or fragment thereof); (3) a set of heavy and lightchain variable region framework surface exposed amino acid residues thatis most closely identical to the set of rodent surface exposed aminoacid residues is identified; and (4) the set of heavy and light chainvariable region framework surface exposed amino acid residues defined instep (2) is substituted with the set of heavy and light chain variableregion framework surface exposed amino acid residues identified in step(3), except for those amino acid residues that are within about 5 Å ofany atom of any residue of a CDR of the, for example, rodent antibody,to yield a humanized, such as, a rodent antibody retaining bindingspecificity.

Antibodies can be humanized by a variety of other techniques includingCDR grafting (EPO 0 239 400; WO 91/09967; and U.S. Pat. Nos. 5,530,101and 5,585,089), veneering or resurfacing (EPO 0 592 106; EPO 0 519 596;Padlan, 1991, Molec Imm 28(4/5):489-498; Studnicka et al., 1994, ProtEng 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973) and chainshuffling (U.S. Pat. No. 5,565,332). Human antibodies can be made by avariety of methods known in the art including, but not limited to, phagedisplay methods, see U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806 and5,814,318; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735 and WO 91/10741, using transgenic animals, such asrodents (Amgen, Kirin and Merdarex mice), using chimeric cells and soon.

“Antibody homolog” or “homolog” refers to any molecule whichspecifically binds extended Type I glycosphingolipid as taught herein.Thus, an antibody homolog includes native or recombinant antibody,whether modified or not, portions of antibodies that retain thebiological properties of interest, such as binding extended Type Iglycosphingolipid, such as an F_(ab) or F_(v) molecule, a single chainantibody, a polypeptide carrying one or more CDR regions and so on. Theamino acid sequence of the homolog need not be identical to that of thenaturally occurring antibody but can be altered or modified to carrysubstitute amino acids, inserted amino acids, deleted amino acids, aminoacids other than the twenty normally found in proteins and so on toobtain a polypeptide with enhanced or other beneficial properties.

Antibodies with homologous sequences are those antibodies with aminoacid sequences that have sequence homology with the amino acid sequenceof an extended Type I glycosphingolipid antibody of the presentinvention. Preferably, homology is with the amino acid sequence of thevariable regions of an antibody of the present invention. “Sequencehomology” as applied to an amino acid sequence herein is defined as asequence with at least about 90%, 91%, 92%, 93%, 94% or more sequencehomology, and more preferably at least about 95%, 96%, 97%, 98% or 99%sequence homology to another amino acid sequence, as determined, forexample, by the FASTA search method in accordance with Pearson & Lipman,Proc Natl Acad Sci USA 85, 2444-2448 (1988).

A chimeric antibody, as taught hereinabove, is one with differentportions of an antibody derived from different sources, such asdifferent antibodies, different classes of antibody, different animalspecies, for example, an antibody having a variable region derived froma murine monoclonal antibody paired with a human immunoglobulin constantregion and so on. Thus, a humanized antibody is a species of chimericantibody. Methods for producing chimeric antibodies are known in theart, see, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986,BioTechniques 4:214; Gillies et al., 1989, J Immunol Methods125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397.

Artificial antibodies include scFv fragments, chimeric antibodies,diabodies, triabodies, tetrabodies and mru (see reviews by Winter &Milstein, 1991, Nature 349:293-299; and Hudson, 1999, Curr Opin Imm11:548-557), each with antigen-binding or epitope-binding ability. Inthe single chain F_(v) fragment (scF_(v)), the V_(H) and V_(L) domainsof an antibody are linked by a flexible peptide. Typically, the linkeris a peptide of about 15 amino acids. If the linker is much smaller, forexample, 5 amino acids, diabodies are formed, which are bivalent scFvdimers. If the linker is reduced to less than three amino acid residues,trimeric and tetrameric structures are formed that are called triabodiesand tetrabodies, respectively. The smallest binding unit of an antibodyis a CDR, for example, CDR3 of the heavy chain which has sufficientspecific recognition and binding capacity. Such a fragment is called amolecular recognition unit or mm. Several such Innis can be linkedtogether with short linker peptides, therefore forming an artificialbinding protein with higher avidity than a single mru.

Also included within the scope of the invention are functionalequivalents of an antibody of interest. The term “functionalequivalents” includes antibodies with homologous sequences, antibodyhomologs, chimeric antibodies, antibody variants, antibody derivatives,artificial antibodies and modified antibodies, for example, wherein eachfunctional equivalent is defined by the ability to bind to extended TypeI glycosphingolipid. The skilled artisan will understand that there isan overlap in the group of molecules termed “antibody fragments” and thegroup termed “functional equivalents.” Methods of producing functionalequivalents which retain extended Type I glycosphingolipid bindingability are known to the person skilled in the art and are disclosed,for example, in WO 93/21319, EPO No. 239,400, WO 89/09622, EPO No.338,745 and EPO No. 332,424.

The functional equivalents of the present application also includemodified antibodies, e.g., antibodies modified by the covalentattachment of any type of molecule to the antibody. For example,modified antibodies include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, deamidation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand, linkage to a toxinor cytotoxic moiety or other protein etc. The covalent attachment neednot yield an antibody that is immune from generating an anti-idiotypicresponse. The modifications may be achieved by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis, chemical conjugation etc.Additionally, the modified antibodies may contain one or morenon-classical amino acids.

Many techniques are available to one of ordinary skill in the art whichpermit the optimization of binding affinity. Typically, the techniquesinvolve substitution of various amino acid residues at the site ofinterest, followed by a screening analysis of binding affinity of themutant polypeptide for the cognate antigen or epitope.

Once the antibody is identified and isolated, it is often useful togenerate a variant antibody or mutant, or mutein, wherein one or moreamino acid residues are altered, for example, in one or more of thehypervariable regions of the antibody. Alternatively, or in addition,one or more alterations (e.g., substitutions) of framework residues maybe introduced in the antibody where these result in an improvement inthe binding affinity of the antibody mutant for extended Type Iglycosphingolipid.

Examples of framework region residues that can be modified include thosewhich non-covalently bind antigen directly (Amit et al., Science233:747-753 (1986)); interact with/affect the conformation of a CDR(Chothia et al., J. Mol. Biol. 196:901-917 (1987)); and/or participatein the V_(L)-V_(H) interface (EP 239 400). In certain embodiments,modification of one or more of such framework region residues results inan enhancement of the binding affinity of the antibody for the cognateantigen. For example, from about one to about five framework residuesmay be altered in the particular embodiment of the invention. Sometimes,that may be sufficient to yield an antibody mutant suitable for use inpreclinical trials, even where none of the hypervariable region residueshave been altered. Normally, however, the antibody mutant can compriseone or more hypervariable region alteration(s). The constant regionsalso can be altered to obtain desirable or more desirable effectorproperties.

The hypervariable region residues which are altered may be changedrandomly, especially where the starting binding affinity of the parentantibody is such that randomly-produced antibody mutants can be readilyscreened for altered binding in an assay as taught herein.

One procedure for obtaining antibody mutants, such as, CDR mutants, is“alanine scanning mutagenesis” (Cunningham & Wells, Science244:1081-1085 (1989); and Cunningham & Wells, Proc Nat. Acad Sci USA84:6434-6437 (1991)). One or more of the hypervariable region residue(s)are replaced by alanine or polyalanine residue(s). Those hypervariableregion residue(s) demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other mutationsat or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. Similarsubstitutions can be attempted with other amino acids, depending on thedesired property of the scanned residues.

A more systematic method for identifying amino acid residues to modifycomprises identifying hypervariable region residues involved in bindingextended Type I glycosphingolipid and those hypervariable regionresidues with little or no involvement with extended Type Iglycosphingolipid binding. An alanine scan of the non-bindinghypervariable region residues is performed, with each ala mutant testedfor enhancing binding to extended Type I glycosphingolipid. In anotherembodiment, those residue(s) significantly involved in binding extendedType glycosphingolipid are selected to be modified. Modification caninvolve deletion of a residue or insertion of one or more residuesadjacent to a residue of interest. However, normally the modificationinvolves substitution of the residue by another amino acid. Aconservative substitution can be a first substitution. If such asubstitution results in a change in biological activity (e.g., bindingaffinity), then another conservative substitution can be made todetermine if more substantial changes are obtained.

Even more substantial modification in an antibody range and presentationof biological properties can be obtained by selecting an amino acid thatdiffers more substantially in properties from that normally resident ata site. Thus, such a substitution can be made while maintaining: (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a sheet or helical conformation; (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain.

For example, the naturally occurring amino acids can be divided intogroups based on common side chain properties:

(1) hydrophobic: methionine (M or met), alanine (A or ala), valine (V orval), leucine (L or leu) and isoleucine (I or ile);

(2) neutral, hydrophilic: cysteine (C or cys), serine (S or ser),threonine (T or thr), asparagine (N or asn) and glutamine (Q or gln);

(3) acidic: aspartic acid (D or asp) and glutamic acid (E or glu);

(4) basic: histidine (H or his), lysine (K or lys) and arginine (R orarg);

(5) residues that influence chain orientation: glycine (G or gly) andproline (P or pro), and

(6) aromatic: tryptophan (W or trp), tyrosine (Y or tyr) andphenylalanine (F or phe).

Non-conservative substitutions can entail exchanging an amino acid withan amino acid from another group. Conservative substitutions can entailexchange of one amino acid for another within a group.

Preferred amino acid substitutions can include those which, for example:(1) reduce susceptibility to proteolysis, (2) reduce susceptibility tooxidation, (3) alter binding affinity and (4) confer or modify otherphysico-chemical or functional properties of such analogs.

Analogs can include various muteins of a sequence other than thenaturally occurring peptide sequence. For example, single or multipleamino acid substitutions (preferably conservative amino acidsubstitutions) may be made in the naturally-occurring sequence(preferably in the portion of the polypeptide outside the domain (s)forming intermolecular contacts. A conservative amino acid substitutionshould not substantially change the structural characteristics of theparent sequence (e.g., a replacement amino acid should not tend to breaka helix that occurs in the parent sequence, or disrupt other types ofsecondary structure that characterizes the parent sequence) unless of achange in the bulk or conformation of the R group or side chain,Proteins, Structures and Molecular Principles (Creighton, ed., W. H.Freeman and Company, New York (1984)); Introduction to Protein Structure(Branden & Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991).

Ordinarily, the antibody mutant with improved biological properties willhave an amino acid sequence having at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the parent anti-human extended Type Iglycosphingolipid antibody, at least 80%, at least 85%, at least 90% andoften at least 95% identity. Identity or similarity with respect toparent antibody sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical (i.e., sameresidue) or similar (i.e., amino acid residue from the same group basedon common side-chain properties, supra) with the parent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity.

Alternatively, antibody mutants can be generated by systematic mutationof the FR and CDR regions of the heavy and light chains, or the F_(c)region of the anti-extended Type I glycosphingolipid antibody.

Another procedure for generating antibody mutants involves the use ofaffinity maturation using phage display (Hawkins et al., J Mot Biol254:889-896 (1992) and Lowman et al., Biochemistry 30(45):10832-10838(1991)). Bacteriophage coat-protein fusions (Smith, Science 228:1315(1985); Scott & Smith; Science 249:386 (1990); Cwirla et al., Proc NatlAcad Sci USA 8:309 (1990); Devlin et al. Science 249:404 (1990); Wells &Lowman, Curt Opin Struct Biol 2:597 (1992); and U.S. Pat. No. 5,223,409)are known to be useful for linking the phenotype of displayed proteinsor peptides to the genotype of bacteriophage particles which encodethem. The F_(ab) domains of antibodies have also been displayed on phage(McCafferty et al., Nature 348: 552 (1990); Barbas et al. Proc Natl AcadSci USA 88:7978 (1991); and Garrard et al. Biotechnol 9:1373 (1991)).

Monovalent phage display consists of displaying a set of proteinvariants as fusions of a bacteriophage coat protein on phage particles(Bass et al., Proteins 8:309 (1990)). Affinity maturation, orimprovement of equilibrium binding affinities of various proteins, haspreviously been achieved through successive application of mutagenesis,monovalent phage display and functional analysis (Lowman & Wells, J MolBiol 234:564 578 (1993); and U.S. Pat. No. 5,534,617), as well as usingthat approach with F_(ab) domains of antibodies (Barbas et al., ProcNatl Acad Sci USA 91:3809 (1994); and Yang et al., J Mol Biol 254:392(1995)).

Libraries of many (for example, 10⁶ or more) protein variants, differingat defined positions in the sequence, can be constructed onbacteriophage particles, each of which contains DNA encoding theparticular protein variant. Thus, several hypervariable region sites(e.g., 6-7 sites) are mutated to generate all possible amino acidsubstitutions at each site. After cycles of affinity purification, usingan immobilized antigen, individual bacteriophage clones are isolated,and the amino acid sequence of the displayed protein is deduced from theDNA.

Following production of the antibody mutant, the biological activity ofthat molecule relative to the parent antibody can be determined astaught herein. As noted above, that may involve determining the bindingaffinity and/or other biological activities or physical properties ofthe antibody. In a preferred embodiment of the invention, a panel ofantibody mutants is prepared and is screened for binding affinity forthe antigen. One or more of the antibody mutants selected from thescreen are optionally subjected to one or more further biologicalactivity assays to confirm that the antibody mutant(s) have new orimproved properties. In preferred embodiments, the antibody mutantretains the ability to bind extended Type I glycosphingolipid with abinding affinity similar to or better/higher than that of the parentantibody.

Alternatively, multivalent phage (McCafferty et al. (1990) Nature348:552-554; and Clackson et al. (1991) Nature 352:624-628) also can beused to express random point mutations (for example, generated by use ofan error-prone DNA polymerase) to generate a library of phage antibodyfragments which then could be screened for affinity to extended Type Iglycosphingolipid, Hawkins et al., (1992) J Mol Biol 254:889-896.

Preferably, during the affinity maturation process, the replicableexpression vector is under tight control of a transcription regulatoryelement, and the culturing conditions are adjusted so the amount ornumber of particles displaying more than one copy of the fusion proteinis less than about 1%. Also preferably, the amount of particlesdisplaying more than one copy of the fusion protein is less than about10% of the amount of particles displaying a single copy of the fusionprotein. Preferably the amount is less than about 20%.

Another equivalent phrase used herein is an antigen binding portion,which relates to that portion of an antibody of interest which binds aType I glycosphingolipid epitope. All of the phrases and terms usedherein to describe various changes that can be made to an originalantibody are considered to fall within the scope of the phrase, “antigenbinding potion.” Hence, for example, an antibody fragment, such as, anF_(ab) molecule, an F_(v), an scAb, an mru, any such functionalfragments, an antibody variant, such as an allele or a moleculecontaining a change in the primary amino acid sequence thereof, aderivative, such as a chimeric or humanized antibody, an analog and soon, including functional equivalents, which include genetically modifiedforms of an antibody of interest, antibody homologs, as describedherein, and so on are included in the phrase antigen binding portion.

The antibody mutant(s) so selected may be subjected to furthermodifications, often depending on the intended use of the antibody. Suchmodifications may involve further alteration of the amino acid sequence,fusion to heterologous polypeptide(s) and/or covalent modifications. Forexample, a cysteine residue not involved in maintaining the properconformation of the antibody mutant may be substituted, generally withserine, to improve the oxidative stability of the molecule and toprevent aberrant cross-linking. Conversely, a cysteine may be added tothe antibody to improve stability (particularly where the antibody is anantibody fragment, such as an F_(v) fragment).

Another type of antibody mutant has an altered glycosylation pattern.That may be achieved by adding or deleting one or more carbohydratemoieties found in the antibody and/or by adding or deleting one or moreglycosylation sites that are not present in the antibody. Glycosylationof antibodies is typically either N-linked to Asn or O-linked to Ser orThr. The tripeptide sequences, asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arecommon recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. N-acetylgalactosamine, galactose,fucose or xylose, for example, are bonded to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine also may be used. Addition or substitution of one ormore serine or threonine residues to the sequence of the originalantibody can enhance the likelihood of O-linked glycosylation.

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance the effectiveness of theantibody. For example, cysteine residue(s) may be introduced in theF_(c) region, thereby allowing interchain disulfide bond formation inthat region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased cell killing mediated bycomplement and antibody-dependent cellular cytotoxicity (ADCC), seeCaron et al., J Exp Med 176:1191-1195 (1992) and Shopes, Immunol148:2918-2922 (1993). Such an antibody derivative or analog also may bemore resistant to degradation in vivo.

Alternatively, an antibody can be engineered which has dual F_(c)regions and may thereby have enhanced complement lysis and ADCCcapabilities, see Stevenson et al., Anti-Cancer Drug Design 3: 219 230(1989).

Covalent modifications of the antibody are included within the scope ofthe invention. Such may be made by chemical synthesis or by enzymatic orchemical cleavage of the antibody, if applicable. Other types ofcovalent modifications of the antibody are introduced into the moleculeby reacting targeted amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor with the N-terminal or C-terminal residue.

Cysteinyl residues can be reacted with α-haloacetates (and correspondingamines), such as chloroacetic acid or chloroacetamide, to yieldcarboxylmethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso can be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercura-4-nitrophenol orchloro-7-nitrobenzo-2-oxa-1,3-diazole, for example.

Histidyl residues can be derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0. p-bromophenacyl bromide also can beused, the reaction is preferably performed in 0.1 M sodium cacodylate atpH 6.0.

Lysinyl and a terminal residues can be reacted with succinic or othercarboxylic acid anhydrides to reverse the charge of the residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters, such as, methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea and 2,4-pentanedione, and the amino acid can betransaminase-catalyzed with glyoxylate.

Arginyl residues can be modified by reaction with one or severalconventional reagents, such as, phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residuesoften requires alkaline reaction conditions. Furthermore, the reagentsmay react with lysine as well as the arginine ε-amino group.

The specific modification of tyrosyl residues can be made with aromaticdiazonium compounds or tetranitromethane. For example, N-acetylimidizoleand tetranitromethane are used to form O-acetyl tyrosyl species and3-nitro derivatives, respectively. Tyrosyl residues can be iodinatedusing ¹²⁵I or ¹³¹I to prepare labeled proteins for use in aradioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) can be modified by reactionwith carbodiimides (R—N═C═C—R′), where R and R′ can be different alkylgroups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively, underneutral or basic conditions. The deamidated form of those residues fallswithin the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of serinyl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (Creighton, Proteins: Structure and Molecular Properties,W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of theN terminal amine and amidation of any C terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling carbohydrates and glycosides to the antibody.Those procedures do not require production of the antibody in a hostcell that has glycosylation capabilities for N-linked or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to: (a) arginine and histidine; (b) free carboxyl groups; (c)free sulfhydryl groups, such as those of cysteine; (d) free hydroxylgroups, such as those of serine, threonine or hydroxyproline; (e)aromatic residues such as those of phenylalanine, tyrosine ortryptophan; or (f) the amide group of glutamine. Such methods aredescribed in WO 87/05330 and in Aplin & Wriston, CRC Crit Rev Biochem,pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylation, forexample, can require exposure of the antibody to the compound,trifluoromethanesulfonic acid, or an equivalent compound, resulting incleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described, for example, inHakimuddin et al., Arch Biochem Biophys 259:52 (1987) and in Edge etal., Anal Biochem 118:131 (1981). Enzymatic cleavage of carbohydratemoieties on antibodies can be achieved by any of a variety ofendoglycosidases and exoglycosidases as described, for example, inThotakura et al., Meth Enzymol 138:350 (1987).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol or polyoxylalkylenes, in themanner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

Functional equivalents may be produced by interchanging different CDR'sof different antibody chains within a framework or a composite FRderived from plural antibodies. Thus, for example, different classes ofantibody are possible for a given set of CDRs by substitution ofdifferent heavy chains, for example, IgG₁₋₄, IgM, IgA₁₋₂ or IgD, toyield differing extended Type I glycosphingolipid antibody types andisotypes. Similarly, artificial antibodies within the scope of theinvention may be produced by embedding a given set of CDR's within anentirely synthetic framework.

The antibody fragments and functional equivalents of the presentinvention encompass those molecules with a detectable degree of specificbinding to extended Type I glycosphingolipid. A detectable degree ofbinding includes all values in the range of at least 10-100%, preferablyat least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90%,95% or 99% of the binding ability of an antibody of interest. Alsoincluded are equivalents with an affinity greater than 100% that of anantibody of interest.

The CDR's generally are of importance for epitope recognition andantibody binding. However, changes may be made to residues that comprisethe CDR's without interfering with the ability of the antibody torecognize and to bind the cognate epitope. For example, changes that donot impact epitope recognition, yet increase the binding affinity of theantibody for the epitope, may be made. Several studies have surveyed theeffects of introducing one or more amino acid changes at variouspositions in the sequence of an antibody, based on the knowledge of theprimary antibody sequence, on the properties thereof, such as, bindingand level of expression (Yang et al., 1995, J Mol Biol 254:392-403;Rader et al., 1998, Proc Natl Acad Sci USA 95:8910-8915; and Vaughan etal., 1998, Nature Biotechnology 16, 535-539).

Thus, equivalents of an antibody of interest can be generated bychanging the sequences of the heavy and light chain genes in the CDR1,CDR2 and/or CDR3, or in the framework regions, using methods such asoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling, amino acid modification ormutator-strains of E. coli (Vaughan et al., 1998, Nat Biotech16:535-539; and Adey et al., 1996, Chap. 16, pp. 277-291, in PhageDisplay of Peptides and Proteins, eds. Kay et al., Academic Press), forexample. The methods of changing the nucleic acid sequence of theprimary antibody can result in antibodies with improved affinity (Gramet al., 1992, Proc Natl Acad Sci USA 89:3576-3580; Boder et al., 2000,Proc Natl Acad Sci USA 97:10701-10705; Davies R. Riechmann, 1996,Immunotech 2:169-179; Thompson et al., 1996, J Mol Biol 256:77-88; Shortet al., 2002, J Biol Chem 277:16365-16370; and Furukawa et al., 2001, JBiol Chem 276:27622-27628).

Repeated cycles of “polypeptide selection” can be used to select forhigher affinity binding by, for example, the selection of multiple aminoacid changes which are selected by multiple selection of cycles.Following a first round of selection, involving a first region ofselection of amino acids in the ligand or antibody polypeptide,additional rounds of selection in other regions or amino acids of theligand are conducted. The cycles of selection are repeated until thedesired affinity properties are achieved.

Improved antibodies also include those antibodies having improvedcharacteristics that are prepared by the standard techniques of animalimmunization, hybridoma formation and selection for antibodies withspecific characteristics.

“Antagonist” refers to a molecule capable of inhibiting one or morebiological activities associated with extended Type I glycosphingolipid.Antagonists may interfere with the maintenance and the growth of a cellexpensing a Type I glycosphingolipid. All points of intervention by anantagonist are considered equivalent for purposes of the instantinvention. Thus, included within the scope of the invention areantagonists, e.g., neutralizing antibodies that bind to extended Type Iglycosphingolipid.

“Agonist” refers to an antibody, an antibody fragment, a conjugate andso on, which activates one or more biological activities of extendedType I glycosphingolipid or a cell expressing same. Agonists can act asa mitogen of cells expressing a Type I glycosphingolipid. All points ofintervention by an agonist shall be considered equivalent for purposesof the instant invention. Thus, included within the scope of theinvention are antibodies that bind to extended Type glycosphingolipidand enhance an activity, such as, differentiation, for example.

The terms “cell,” “cell line,” and “cell culture” include progenythereof. It is also understood that all progeny may not be preciselyidentical, such as, in DNA content, due to deliberate or inadvertentmutation. Variant progeny that have the same function or biologicalproperty of interest, as screened for in the original cell, are includedin the scope of the invention. The “host cells” used in the presentinvention generally are prokaryotic or eukaryotic hosts, selected as adesign choice.

“Transformation” of a cellular organism, cell or cell line with anucleic acid means introducing a nucleic acid into the target cell sothat the nucleic acid is replicable, either as an extrachromosomalelement or by chromosomal integration, and, optionally, expressed.“Transfection” of a cell or organism with a nucleic acid refers to thetaking up of the nucleic acid, e.g., an expression vector, by the cellor organism whether or not any coding sequences are in fact expressed.The terms “transfected host cell” and “transformed” refer to a cell inwhich a nucleic acid was introduced. Typical prokaryotic host cellsinclude various strains of E. coli. Typical eukaryotic host cells aremammal cells, such as Chinese hamster ovary, or cells of human origin.The introduced nucleic acid sequence may be from the same species as thehost cell or of a different species from the host cell, or may be ahybrid nucleic acid sequence, containing some foreign and somehomologous nucleic acids. Transformation can also occur by transductionor infection with virus-derived elements or carriers.

The term “vector” means a nucleic acid construct, a carrier, containinga nucleic acid, the transgene, the foreign gene or the gene of interest,which can be operably linked to suitable control sequences forexpression of the transgene in a suitable host. Such control sequencesinclude, for example, a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites and sequences which control thetermination of transcription and translation. The vector may be aplasmid, a phage particle or just a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may in some instances, integrateinto a host cell genome or other nucleic acid. In the presentspecification, “plasmid” and “vector” are used interchangeably, as aplasmid is a commonly used form of vector. However, the invention isintended to include such other forms of vectors which serve equivalentcarrier function as and which are, or become, known in the art, such as,viruses, phagemids, transposons, synthetic molecules that carry nucleicacids, liposomes and the like.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including human, domestic and farm animals, nonhuman primates,and zoo, sports or pet animals, such as dogs, horses, cats, cows etc.

The antibodies of interest can be screened or can be used in an assay asdescribed herein or as known in the art. Often, such assays require areagent to be detectable, that is, for example, labeled. The word“label” when used herein refers to a detectable compound or compositionwhich can be conjugated directly or indirectly to a molecule or protein,e.g., an antibody. The label may itself be detectable (e.g.,radioisotope labels, particles or fluorescent labels) or may be aninstrument to obtain a detectable signal, such as, in the case of anenzymatic label, may catalyze a chemical alteration of a substratecompound or composition which then is detectable.

As used herein, “solid phase” means a matrix to which an entity ormolecule, such as, the antibody of the instant invention, can adhere orbind. Example of solid phases encompassed herein include those formedpartially or entirely of glass (e.g., controlled pore glass),polysaccharides (e.g., agarose), plastics, polypropylenes,polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others can be used in apurification column (e.g., an affinity chromatography column). Thus, thesolid phase can be a paper, a bead, a plastic, a chip and so on, can bemade from a variety of materials, such as nitrocellulose, agarose,polystyrene, polypropylene, silicon and so on, and can be in a varietyof configurations.

Cells expressing extended Type I glycosphingolipid or glycans thereof,such as cell membrane preparations, as well as purified extended Type Iglycosphingolipid can be used as immunogens for generating antibodies ofinterest. The immunogen can be obtained or isolated from natural sourcesor can be made synthesized enzymatically or chemically. Whole cells,such as extended Type I glycosphingolipid expressing cells, cellsderived from a natural source or from cancers, such as cancer celllines, can be used. Cells that overexpress extended Type Iglycosphingolipid may be used as the immunogen for making the antibodiesof interest. Also, membrane preparations carrying extended Type Iglycosphingolipid can be used, as known in the art. Such cells andportions thereof can be used as the antigen source in a diagnosticassay.

Nucleic acid molecules encoding amino acid sequence mutants can beprepared by a variety of methods known in the art. The methods include,but are not limited to, oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis and cassette mutagenesis of an earlierprepared mutant or a non-mutant version of the molecule of interest,(see, for example, Kunkel, Proc Natl Acad Sci USA 82:488 (1985)).

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention, a single chain antibody of the invention oran antibody mutein of the invention) includes construction of anexpression vector containing a polynucleotide that encodes the antibodyor a fragment of the antibody as described herein. Once a polynucleotideencoding an antibody molecule has been obtained, the vector for theproduction of the antibody may be produced by recombinant DNA technologyas known in the art. An expression vector is constructed containingantibody coding sequences and appropriate transcriptional andtranslational control signals. The methods include, for example, invitro recombinant DNA techniques, synthetic techniques and in vivogenetic recombination.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells then are cultured by conventionaltechniques to produce an antibody, or fragment, of the invention. In oneaspect of the invention, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed herein.

A variety of host/expression vector systems may be utilized to expressthe antibody molecules of the invention. Such expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ.Bacterial cells, such as E. coli, and eukaryotic cells are commonly usedfor the expression of a recombinant antibody molecule, especially forthe expression of whole recombinant antibody molecule. For example,mammal cells such as CHO cells, in conjunction with a vector, such asone carrying the major intermediate early gene promoter element fromhuman cytomegalovirus, are an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); and Cockett et al., Bio/Technology8:2 (1990)). Plants and plant cell culture, insect cells and so on alsocan be used to make the proteins of interest, as known in the art.

In addition, a host cell is chosen which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products may be important for thefunction of the protein. Different host cells can have the particularcharacteristic and specific mechanisms for the desiredpost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the expressed antibody ofinterest. Hence, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript, glycosylationand phosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, COS, 293, 3T3 or myelomacells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer sequences, transcription terminators,polyadenylation sites etc.) and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor one to two days in an enriched media, and then are moved to aselective medium. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into a chromosome and be expanded into a cell line.Alternatively, an extrachromosomal element can be maintained in thecells under selection. Such engineered cell lines not only are usefulfor antibody production but are useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule.

A number of selection systems may be used, including but not limited tousing the Herpes simplex virus thymidine kinase (Wigler et al.; Cell11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska et al., Proc Natl Acad Sci USA 48:202 (1992)), glutamatesynthase, in the presence of methionine sulfoximide (Adv Drug Del Rev58, 671, 2006 and see the website or literature of Lonza Group Ltd.) andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes in tk, hgprt or aprt cells, respectively. Also, antimetaboliteresistance can be used as the basis of selection for the followinggenes: dhfr, which confers resistance to methotrexate (Wigler et al.,Proc Natl Acad Sci USA 77:357 (1980); O'Hare et al., Proc Natl Acad SciUSA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid(Mulligan et al., Proc Natl Acad Sci USA 78:2072 (1981)); neo, whichconfers resistance to the aminoglycoside, G-418 (Wu et al., Biotherapy3:87 (1991)); and hygro, which confers resistance to hygromycin(Santerre et al., Gene 30:147 (1984)). Methods known in the art ofrecombinant DNA technology may be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al., eds., Current Protocols in Molecular Biology, JohnWiley & Sons (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press (1990); Dracopoli et al., eds.,Current Protocols in Human Genetics, John Wiley & Sons (1994); andColberre-Garapin et al., J Mol Biol 150:1 (1981).

The expression levels of an antibody molecule can be increased by vectoramplification (for example, see Bebbington et al., in DNA Cloning, Vol.3. Academic Press (1987)). When a marker in the vector system expressingantibody is amplifiable, an increase in the level of inhibitor presentin the culture will increase the number of copies of the marker gene.Since the amplified region is associated with the antibody gene,production of the antibody will also increase (Crouse et al., Mol CellBiol 3:257 (1983)).

The host cell may be co-transfected with two or more expression vectorsof the invention, for example, the first vector encoding a heavychain-derived polypeptide and the second vector encoding a lightchain-derived polypeptide. The two vectors may contain identicalselectable markers which enable equal expression of heavy and lightchain polypeptides. Alternatively, a single vector may be used whichencodes, and is capable of expressing, both heavy and light chainpolypeptides. In such situations, the light chain can be placed beforethe heavy chain to avoid an excess of toxic free heavy chain (Proudfoot,Nature 322:52 (1986); and Kohler, Proc Natl Acad Sci USA 77:2197(1980)). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for extended Type Iglycosphingolipid after Protein A and size-exclusion chromatography, andso on), centrifugation, differential solubility or by any other standardtechnique for the purification of proteins. In addition, the antibodiesof the instant invention or fragments thereof can be fused toheterologous polypeptide sequences described herein or otherwise knownin the art, to facilitate purification.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Thus, a purified extended Type I structure canbe used as antigen, optionally, with an adjuvant, such as, complete orincomplete Freund's adjuvant. The antibodies of the present inventionmay comprise polyclonal antibodies, although because of the modificationof antibodies to optimize use in human, as well as to optimize the useof the antibody per se, monoclonal antibodies are preferred because ofease of production and manipulation of particular proteins. Methods ofpreparing polyclonal antibodies are known to the skilled artisan (Harlowet al., Antibodies: a Laboratory Manual, Cold Spring Harbor LaboratoryPress, 2nd ed. (1988)).

For example, an immunogen, as exemplified herein, may be administered tovarious host animals including, but not limited to, rabbits, mice,camelids, rats etc., to induce the production of serum containingpolyclonal antibodies specific for extended Type I glycosphingolipid.The administration of the immunogen may entail one or more injections ofan immunizing agent and, if desired, an adjuvant. Various adjuvants maybe used to increase the immunological response, depending on the hostspecies, and include, but are not limited to, Freund's (complete andincomplete), mineral oil, gels, alum (aluminum hydroxide), surfaceactive substances, such as lysolecithin, pluronic polyols, polyanions,peptides, oil emulsions, keyhole limpet hemocyanins (KLH), dinitrophenoland potentially useful human adjuvants, such as BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Additional examples ofadjuvants which may be employed include the MPL-TDM adjuvant(monophosphoryl lipid A, synthetic trehalose dicorynomycolate).Immunization protocols are well known in the art and may be performed byany method that elicit an immune response in the animal host chosen.Thus, various administration routes can be used over various timeperiods as a design choice.

Typically, the immunogen (with or without adjuvant) is injected into themammal by multiple subcutaneous or intraperitoneal injections, orintramuscularly or intravenously. In certain circumstances, whole cellsexpressing extended Type I glycosphingolipid can be used. Depending onthe nature of the immunogen (i.e., percent hydrophobicity, percenthydrophilicity, stability, net charge, isoelectric point etc.), theextended Type I glycosphingolipid or portion thereof may be modified orconjugated to be immunogenic or more immunogenic in the animal, such asa mammal, being immunized. For example, extended Type Iglycosphingolipid or a portion thereof can be conjugated to a carrier.The conjugation includes either chemical conjugation by derivatizingactive chemical functional groups on either or both the immunogen andthe immunogenic protein to be conjugated such that a covalent bond isformed or other methods known to the skilled artisan. Examples of suchcarriers or immunogenic proteins include, but are not limited to, KLH,ovalbumin, serum albumin, bovine thyroglobulin, soybean trypsininhibitor and promiscuous T helper peptides. Various adjuvants may beused to increase the immunological response as described above.

Once a suitable preparation is obtained, it is possible to isolateparticular antibodies from the plural antibodies by known separationtechniques, such as affinity chromatography, panning, absorption and soon. In that way, an individual antibody species can be obtained forfurther study, for example, sequencing to obtain the amino acidsequences of one or more CDRs.

The antibodies of the present invention preferably comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomatechnology, such as described by Kohler et al., Nature 256:495 (1975);U.S. Pat. No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 2nd ed. (1988); and Hammerling etal., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier (1981),recombinant DNA methods, for example, making and using transfectomas, orother methods known to the artisan. Other examples of methods which maybe employed for producing monoclonal antibodies include, but are notlimited to, the human B-cell hybridoma technique (Kosbor et al.,Immunology Today 4:72 (1983); and Cole et al., Proc Natl Acad Sci USA80:2026 (1983)), and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss(1985)). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA and IgD, and any subclass thereof. The hybridomaproducing the mAb of the invention may be cultivated in vitro or invivo.

In the hybridoma model, a host such as a mouse, a humanized mouse, atransgenic mouse with human immune system genes, horse, sheep, hamster,rabbit, rat, camel or any other appropriate host animal, is immunized toelicit lymphocytes that produce or are capable of producing antibodiesthat specifically bind to extended Type I glycosphingolipid.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)).

Generally, in making antibody-producing hybridomas, either peripheralblood lymphocytes (“PBLs”) are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine or human origin. Typically, a rat or mouse myeloma cell line isemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme, hypoxanthine guanine phosphoribosyltransferase (HGPRT or

Preferred immortalized cell lines are those that fuse efficiently,support stable high level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium, such as HATmedium. Among the myeloma cell lines are murine myeloma lines, such asthose derived from the MOPC-21 and MPC-11 mouse tumors available fromthe Salk Institute Cell Distribution Center, San Diego, Calif. andSP2/0, FO or X63-Ag8-653 cells available from the American Type CultureCollection, Manassas, Va. The mouse myeloma cell line NSO also may beused (European Collection of Cell Cultures, Salisbury, Wilshire, UK).

Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, JImmunol 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc, pp. 51-63(1987)).

Another alternative is to use electrical fusion rather than chemicalfusion to form hybridomas. Instead of chemical fusion, a B cell can beimmortalized using, for example, Epstein Barr Virus or anothertransforming gene, see, e.g., Zurawaki et al., in Monoclonal Antibodies,ed., Kennett et al., Plenum Press, pp. 19-33. (1980). Transgenic miceexpressing immunoglobulins and severe combined immunodeficient (SCID)mice transplanted with human B lymphocytes also can be used.

The culture medium in which hybridoma cells are grown is assayed forproduction of monoclonal antibodies directed against extended Typeglycosphingolipid. The binding specificity of monoclonal antibodiesproduced by hybridoma cells may be determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA),fluorocytometric analysis (FACS) or enzyme-linked immunosorbent assay(ELISA). Such techniques are known in the art and are within the skillof the artisan. Also, the Biacore system can be used, as known in theart. The binding affinity of the monoclonal antibody to extended Type Iglycosphingolipid can, for example, be determined by a Scatchardanalysis (Munson et al., Anal Biochem 107:220 (1980)).

After hybridoma cells that produce antibodies of the desiredspecificity, affinity and/or activity are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,pp. 59-103 (1986)). Suitable culture medium includes, for example,Dulbecco's Modified Eagle's Medium (D-MEM) or RPMI-1640. In addition,the hybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated or isolated from the culture medium, ascites fluid or serum byconventional immunoglobulin purification procedures such as, forexample, protein A Sepharose, protein G Sepharose, hydroxylapatitechromatography, gel exclusion chromatography, gel electrophoresis,dialysis or affinity chromatography.

A variety of methods exist in the art for the production of monoclonalantibodies and thus, the invention is not limited to their soleproduction in hybridomas. For example, the monoclonal antibodies may bemade by recombinant DNA methods, such as those described in U.S. Pat.No. 4,816,567. Alternatively, human antibodies can be obtained fromtransgenic animals, such as the KM mouse, discussed above. In thatcontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, viral or prokaryotic clone.

Thus, using human cancer cells known to express an extended Type chainstructure, such as Colo205 cells, or an extended Type I chain containingcompound, such as glycosphingolipid, such as Le^(b)/Le^(a), as antigen,inbred or transgenic mice are immunized and boosted as known in the art.Spleens were obtained, cells fused to myeloma cells and hybridomas madeand cultured. Cell supernatants were screened by ELISA using, forexample, Le^(b)/Le^(a) as the capture reagent. Positive clones wereamplified. IMH2 is an example of a mouse I_(g)G₃ monoclonal antibodythat binds specifically to an extended Type I chain structure.

Using a transgenic mouse model, human antibodies can be produced byimmunizing the transgenic mice with a extended Type I chain immunogen.Such antibodies can be generated on a fee basis, for example, byMedarex, N.J. and Amgen, Calif. Using the KM mouse, the GNX-8 (IgG₁)monoclonal antibody was selected for further characterization and use.

GNX-8 is a cytotoxic antibody. In assays using Colo205 colon cancercells as targets, GNX-8 lysed the cancer cell line cells, and at leastat 50 μg/ml, the antibody lysed all cells in the culture. GNX-8 does notbind to RBCs. The antibody binds to colorectal cancer cells, breastcancer cells and lung cancer cells. Unlike IMH2, GNX-8 does not bind toLe^(y)-Le^(x) or Le^(y).

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies, or such chainsfrom human, humanized or other sources) (Innis et al. in PCR Protocols.A Guide to Methods and Applications, Academic (1990) and Sanger et al.,Proc Natl Acad Sci 74:5463 (1977)). The hybridoma cells can serve as thesource of such DNA.

Once isolated, the DNA may be placed into expression vectors, which arethen transfected into host cells such as E. coli cells, NSO cells, COScells, Chinese hamster ovary (CHO) cells or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. The DNA also may bemodified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the homologous murinesequences (U.S. Pat. No. 4,816,567; and Morrison et al., Proc Natl AcadSci USA 81:6851 (1984)) or by covalently joining to the immunoglobulincoding sequence, all or part of the coding sequence of anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneextended Type I glycosphingolipid-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the F_(c) region so as to prevent heavy chain cross-linking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to preventcross-linking.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. Traditionally, those fragments are derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,J Biochem Biophys Methods 24:107 (1992); and Brennan et al., Science229:81 (1985)). For example, F_(ab) and F_((ab′)2) fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes, such as, papain (to produce F_(ab) fragments)or pepsin (to produce F_((ab′)2) fragments). F_((ab′)2) fragmentscontain the variable region, the light chain constant region and theC_(H1) domain of the heavy chain. However, those fragments can beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from an antibody phage library. Alternatively,F_((ab′)2)-SH fragments can be directly recovered from E. coli andchemically coupled to form F_((ab′)2) fragments (Carter et al.,Bio/Technology 10:163 (1992). According to another approach, F_((ab′)2)fragments can be isolated directly from recombinant host cell culture.Other techniques for the production of antibody fragments will beapparent to the skilled practitioner. In other embodiments, the antibodyof choice is a single chain F, fragment (F_(v)), see, for example, WO93/16185.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanizedor human antibodies. Methods for producing chimeric antibodies are knownin the art, see e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986);

Humanized antibodies are derived from antibody molecules generated in anon-human species that bind extended Type I glycosphingolipid whereinone or more CDR's therefrom are inserted into she FR regions from ahuman immunoglobulin molecule. Antibodies can be humanized using avariety of techniques known in the art including, for example, CDRgrafting (EPO 239,400; WO 91/09967; and U.S. Pat. Nos. 5,225,539;5,530,101; and 5,585,089), veneering or resurfacing (EPO 592,106; EPO519,596; Padlan, Molecular Immunology 28:489 (1991); Studnicka et al.,Protein Engineering 7:805 (1994); and Roguska et al., Proc Natl Acad SciUSA 91:969 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

A humanized antibody has one or more amino acid residues from a sourcethat is non-human. The non-human amino acid residues are often referredto as “import” residues, which are typically taken from an “import”variable domain. Humanization can be essentially performed following themethods of Winter et al. (Jones et al., Nature 321:522 (1986); Riechmannet al., Nature 332:323 (1988); and Verhoeyen et al., Science 239:1534(1988)), by substituting non-human CDR's or portions of CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possible some FR residues aresubstituted from analogous sites in rodent antibodies. The heavy chainconstant region and hinge region can be from any class or subclass toobtain a desired effect, such as a particular effector function.

Often, framework residues in the human framework regions can besubstituted with the corresponding residue from the CDR donor antibodyto alter, and possibly improve, antigen binding. The frameworksubstitutions are identified by methods known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding

It is further preferable that humanized antibodies retain high affinityfor extended Type I glycosphingolipid, and retain or acquire otherfavorable biological properties. Thus, humanized antibodies can beprepared by a process by analyzing the parental sequences and variousconceptual humanized antibody derivatives using three-dimensional modelsof the parental and humanized sequences. The hypotheticalthree-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthe displays permits analysis of the likely role of certain residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind extended Type I glycosphingolipid. In that way,FR residues can be selected and combined from the recipient and importsequences so that the desired antibody characteristic, such as increasedaffinity for the target antigen, is maximized, although it is the CDRresidues that directly and most substantially influence extended Type Iglycosphingolipid binding. The CDR regions also can be modified tocontain one or more amino acids that vary from that obtained from theparent antibody from which the CDR was obtained, to provide enhanced ordifferent properties of interest, such as binding of greater affinity orgreater avidity, for example.

Certain portions of the constant regions of antibody can be manipulatedand changed to provide antibody homologs, derivatives, fragments and thelike with properties different from or better than that observed in theparent antibody. Thus, for example, many IgG4 antibodies form intrachaindisulfide bonds near the hinge region. The intrachain bond candestabilize the parent bivalent molecule forming monovalent moleculescomprising a heavy chain with the associated light chain. Such moleculescan reassociate, but, on a random basis.

Another set of amino acids suitable for modification include amino acidsin the area of the hinge which impact antibody functions, such as,binding of a molecule containing a heavy chain with binding to the F_(c)receptor and internalization of bound antibody. Such amino acidsinclude, in IgG1 molecules, residues from about 233 to about 237(Glu-Leu-Leu-Gly-Gly, SEQ ID NO:1); from about 252 to about 256(Met-Ile-Ser-Arg-Thr, SEQ ID NO:2) and from about 318 (Glu) to about 331(Pro), including, for example, Lys₃₂₀, Lys₃₂₂ and Pro₃₂₉.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences, see, U.S. Pat. Nos. 4,444,887 and 4,716,111; and WO 98/46645,WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO91/10741. The techniques of Cole et al. and Boerder et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985); andBoerner et al., J Immunol 147:86 (1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichalso express certain human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region and diversityregion may be introduced into mouse embryonic stem cells, in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be treated so as to be non-functionalseparately or simultaneously with the introduction of the humanimmunoglobulin loci by homologous recombination. In particular,homozygous deletion of the JH region prevents endogenous antibodyproduction. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies, see, e.g., Jakobovitis et al., Proc Natl Acad Sci USA90:2551 (1993); Jakobovitis et al., Nature 362:255 (1993); Bruggermannet al., Year in Immunol 7:33 (1993); and Duchosal et al., Nature 355:258(1992)).

The transgenic mice are immunized in the normal fashion with an extendedType I glycosphingolipid, e.g., all or a portion of extended Type Iglycosphingolipid, or a membrane preparation containing same. Monoclonalantibodies directed against extended Type I glycosphingolipid can beobtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgM and IgE antibodies. For an overview, see Lonberg et al., Int RevImmunol 13:65-93 (1995). For a discussion of producing human antibodiesand human monoclonal antibodies and protocols for producing suchantibodies, see, e.g., WO 98/24893; WO 92/01047; WO 96/34096; and WO96/33735; EPO No. 0 598 877; and U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598. In addition, companies such as Amgen (Fremont,Calif.), Genpharm (San Jose, Calif.) and Medarex, Inc. (Princeton, N.J.)can be engaged to provide human antibodies directed against extendedType I glycosphingolipid using technology similar to that describedabove.

Also, human mAbs could be made by immunizing mice transplanted withhuman peripheral blood leukocytes, splenocytes or bone marrow (e.g.,trioma technique of XTL Biopharmaceuticals, Israel).

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thatapproach, a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., Bio/Technology 12:899(1988)).

When using recombinant techniques, the antibody variant can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody variant is produced intracellularly, as a firststep, the

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis and affinity chromatography. The suitability of protein A orprotein G as an affinity ligand depends on the species and isotype of animmunoglobulin F_(c) domain that is present in the antibody variant.Protein A can be used to purify antibodies that are based on human IgG1,IgG2 or IgG4 heavy chains (Lindmark et al., J Immunol Meth 62:1 (1983)).Protein G can be used for mouse isotypes and for human IgG3 (Guss etal., EMBO J. 5:1567 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices, such as controlled pore glass orpoly(styrenedivinyl)benzene, allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodyvariant comprises a C_(H3) domain, the Bakerbond ABXTM resin (J T Baker;Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification, such as, fractionation on an ion-exchange column,ethanol precipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin agarose chromatography on an anion or cationexchange resin (such as a polyaspartic acid column), chromatofocusing,SDS-PAGE and ammonium sulfate precipitation are also available,depending on the antibody or variant to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody or variant of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH of between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25 M salt).

Further, antibodies of the invention can, in turn, be utilized togenerate anti-idiotype antibodies that “mimic” extended Type Iglycosphingolipid using techniques well known to those skilled in theart (see, e.g., Greenspan et al., FASEB J 7:437 (1989); and Nissinoff, JImmunol 147:2429 (1991)). For example, antibodies which bind to andcompetitively inhibit multimerization and/or binding of a ligand toextended Type I glycosphingolipid can be used to generate anti-idiotypesthat “mimic” extended Type I glycosphingolipid. Such neutralizinganti-idiotypes or F_(ab) fragments of such anti-idiotypes can be used intherapeutic or diagnostic regimens.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies can be monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities isdirected towards extended Type I glycosphingolipid, whereas the otherspecificity may be for any other antigen, such as a cell-surfaceprotein, receptor, receptor subunit, ligand, tissue-specific antigen,viral protein, virally-encoded envelope protein, pharmacologicallyactive agent, such as a drug, bacterially-derived protein, bacterialsurface protein etc.

Methods for making bispecific antibodies are well known. Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy chain/light chain pairs, wherethe two heavy chains have different specificities (Milstein et al.,Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, the hybridomas (quadromas)produce a potential mixture of about ten different antibody molecules,of which only about one might have the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829and in Traunecker et al., EMBO J 10:3655 (1991). Other methods formaking bispecific antibodies are provided in, for example, Kufer et al.,Trends Biotech 22:238-244, 2004.

Antibody variable domains with the desired binding specificities can befused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H2) and C_(H3) regions. It may have thefirst heavy chain constant region (C_(H1)) containing the site necessaryfor light chain binding present in at least one of the fusions. DNA'sencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth Enzym 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980).It is contemplated that the antibodies may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioester bond. Examplesof suitable reagents for that purpose include iminothiolate andmethyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S.Pat. No. 4,676,980.

In addition, one can generate single domain antibodies to extended TypeI glycosphingolipid. Examples of that technology have been described inWO9425591 for antibodies derived from Camelidae heavy chain Ig, as wellas in US20030130496 describing the isolation of single domain fullyhuman antibodies from phage libraries.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423 (1988);Huston et al., Proc Natl Acad Sci USA 85:5879 (1988); and Ward et al.,Nature 334:544 (1989)) can be practiced. Single chain antibodies areformed by linking the heavy and light chain fragments of the F_(v)region via an amino acid bridge, resulting in a single chainpolypeptide. Techniques for the assembly of functional fragments in E.coli may also be used (Skerra et al., Science 242:1038 (1988)). Singlechain antibodies (“scF_(v)”) and a method of their construction aredescribed in, for example, U.S. Pat. No. 4,946,778. Alternatively,F_(ab) can be constructed and expressed by similar means. All of thewholly and partially human antibodies can be less immunogenic thanwholly murine mAbs, and the fragments and single chain antibodies alsocan be less immunogenic.

The instant invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide. Fused or conjugated antibodies of thepresent invention may be used for ease in purification, see e.g., WO93/21232; EP 439,095; Naramura et al., Immol Lett 39:91 (1994); U.S.Pat. No. 5,474,981; Gillies et al., Proc Natl Acad Sci USA 89:1428(1992); and Fell et al., J Immunol 146:2446 (1991).

The purification can be facilitated by using a recognition marker ortag. For example, the marker can be an amino acid sequence, such as, ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc., Chatsworth, Calif.), among others, many of which arecommercially available, Gentz et al., Proc Natl Acad Sci USA 86:821(1989). Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

Antibodies or antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature 348:552 (1990). Clarkson et al., Nature 352:624 (1991) and Markset al., J Mol Biol 222:581 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Marks et al., Bio/Technology 10:779(1992)), as well as combinatorial infection and in vivo recombination asa strategy for constructing very large phage libraries (Waterhouse etal., Nucl Acids Res 21:2265 (1993)). Thus, the techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

Candidate anti-extended Type I glycosphingolipid antibodies can betested by enzyme-linked immunosorbent assay (ELISA), FACS, Westernimmunoblotting or other immunochemical techniques, as known in the art.Thus, B cells or cells expressing extended Type I glycosphingolipid canbe used to detect antibody binding thereto using a known technique, orsuitable membrane preparations containing extended Type Iglycosphingolipid or portion thereof, or purified or isolated extendedType I chain structures can be adhered to a solid phase and used as acapture element in an assay, configured as a design choice.

To determine whether a particular antibody homolog binds to humanextended Type I glycosphingolipid, any conventional binding assay may beused. Useful extended Type I glycosphingolipid binding assays includeFACS analysis, ELISA assays, radioimmunoassays and the like, whichdetect binding of antibody, and functions resulting therefrom, to humanextended Type I glycosphingolipid. Full-length and soluble forms ofhuman extended Type I glycosphingolipid taught herein are useful in suchassays. The binding of an antibody or homolog to extended Type Iglycosphingolipid, or to soluble fragments thereof, may conveniently bedetected through the use of a second antibody specific forimmunoglobulins of the species from which the antibody or homolog isderived. The second antibody can carry a detectable label or configuredto be detected.

The ability of an antibody or homolog to bind to human extended Type Iglycosphingolipid can be evaluated by testing the ability thereof tobind to human extended Type I glycosphingolipid⁺ cells. Suitableextended Type I glycosphingolipid⁻ cells for use in determining whethera particular antibody or homolog binds to human extended Type Iglycosphingolipid are available mammal tissue culture cells expressingextended Type I glycosphingolipid, such as, on the cell surface.

Binding of the antibody or homolog to the extended Type Iglycosphingolipid⁺ cell can be detected by staining the cells with, forexample, a fluorescently-labeled second antibody specific forimmunoglobulins of the same species from which the antibody homologbeing tested is derived. A fluorescence activated cell sorter (“FACS”)can be used to detect and to quantify any binding, see generally,Shapiro, Practical Flow Cytometry, Alan R. Liss, Inc., New York, N.Y.(1985).

To determine whether a particular antibody or homolog causes nosignificant decrease in the number of circulating extended Type Iglycosphingolipid cells in vivo, the number of circulating extended TypeI glycosphingolipid⁺ cells isolated from a mammal within 24 hours afteradministration of the antibody or homolog to a mammal having normalimmune function is quantified, and compared to the pre-administrationnumber or the number in a control mammal to whom an isotype-matchedantibody or homolog of irrelevant specificity has been administeredinstead of an antibody or homolog of the instant invention.Quantification of extended Type I glycosphingolipid⁺ cells in animalsdosed with an extended Type glycosphingolipid antibody or functionalportion or derivative thereof may be accomplished, for example, bystaining obtained cells with fluorescently-labeled antibodies that bindthe anti-extended Type I glycosphingolipid antibodies, as well aslabeled antibodies specific for T cells and B cells, followed by FACSanalysis.

Antibodies of the instant invention may be described or specified interms of the epitope(s) or portion(s) of extended Type Iglycosphingolipid to which the antibody recognizes or specificallybinds. The epitope(s) may be specified as described herein, e.g., byphysical means, such as mass spectrometry, compositional analysis of thesaccharides, the molecules to which the sugars bind, conformationalepitopes and so on.

Antibodies of the instant invention may also be described or specifiedin terms of cross-reactivity. Antibodies that bind extended Type Iglycosphingolipids, which have at least 95%, at least 90%, at least 85%,at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, atleast 55%, and at least 50% identity (as calculated using methods knownin the art and described herein) to extended Type I glycosphingolipidare also included in the instant invention.

Antibodies of the instant invention also may be described or specifiedin terms of binding affinity to an extended Type I glycosphingolipid ofinterest. Anti-extended Type I glycosphingolipid antibodies may bindwith a K_(D) of less than about 10⁻⁷ M, less than about 10⁻⁶ M, or lessthan about 10⁻⁵ M. Higher binding affinities in an antibody of interestcan be beneficial, such as those with an equilibrium dissociationconstant or K_(D) of from about 10⁸ to about 10⁻¹⁵ M or more, from about10⁻⁸ to about 10⁻¹²M, from about 10⁻⁹ to about 10⁻¹¹ M, or from about10⁻⁸ to about 10⁻¹⁰ M. The invention also provides antibodies thatcompetitively inhibit binding of an antibody to an epitope of theinvention as determined by any method known in the art for determiningcompetitive binding, for example, the immunoassays described herein. Inpreferred embodiments, the antibody competitively inhibits binding tothe epitope by at least 95%, at least 90%, at least 85%, at least 80%,at least 75%, at least 70%, at least 60%, or at least 50%.

The instant invention also includes conjugates comprising an antibody ofinterest. The conjugates comprise two primary components, an antibody ofinterest and a second component, which may be a cell-binding agent, acytotoxic agent, a pharmacologically active agent, a drug and so on.

As used herein, the term “cell-binding agent” refers to an agent thatspecifically recognizes and binds to a molecule on the cell surface.Thus, the cell-binding agent can be one that binds a CD antigen, apathogen antigen, such as a virus antigen, a differentiation antigen, acancer antigen, a cell-specific antigen, a tissue-specific antigen, anIg or Ig-like molecule and so on.

Cell-binding agents may be of any type as presently known, or thatbecome known, and includes peptides, non-peptides, saccharides, nucleicacids, ligands, receptors and so on, or combinations thereof. Thecell-binding agent may be any compound that can bind a cell, either in aspecific or non-specific manner. Generally, the agent can be an antibody(especially monoclonal antibodies), lymphokines, hormones, growthfactors, vitamins, nutrient-transport molecules (such as transferrin),or any other cell-binding molecule or substance.

Other examples of cell-binding agents that can be used include:polyclonal antibodies; monoclonal antibodies; and fragments ofantibodies such as F_(ab), F_(ab′), F_((ab′)2) and F_(v) fragments(Parham, J. Immunol. 131:2895-2902 (1983); Spring et al., J. Immunol.113:470-478 (1974); and Nisonoff et al., Arch. Biochem. Biophys. 89:230-244 (1960)).

The second component also can be a cytotoxic agent. The term “cytotoxicagent” as used herein refers to a substance that reduces or blocks thefunction or growth, of cells and/or causes destruction of cells. Thus,the cytotoxic agent can be a taxol, a maytansinoid, such as, DM1 or DM4,CC-1065 or a CC-1065 analog, a ricin, a drug, mitomycin C and so on. Insome embodiments, the cytotoxic agent, as with any binding agent of aconjugate of the instant invention, is covalently attached, directly orvia a cleavable or non-cleavable linker, to an antibody of interest.

Examples of suitable maytansinoids include maytansinol and maytansinolanalogs. Maytansinoids inhibit microtubule formation and are highlytoxic to mammalian cells.

Examples of suitable maytansinol analogues include those having amodified aromatic ring and those having modifications at otherpositions. Such suitable maytansinoids are disclosed in U.S. Pat. Nos.4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Examples of suitable analogues of maytansinol having a modified aromaticring include: (1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared, forexample, by LAH reduction of ansamytocin P2); (2) C-20-hydroxy (orC-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016)(prepared, for example, by demethylation using Streptomyces orActinomyces or dechlorination using lithium aluminum hydride (LAH)); and(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Examples of suitable analogues of maytansinol having modifications ofother positions include: (1) C-9-SH (U.S. Pat. No. 4,424,219) (preparedby the reaction of maytansinol with H₂S or P₂S₅); (2) C-14-alkoxymethyl(demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598); (3) C-14-hydroxymethyl oracyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No. 4,450,254) (prepared fromNocardia); (4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (preparedby the conversion of maytansinol by Streptomyces); (5) C-15-methoxy(U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewianudiflora); (6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348)(prepared by the demethylation of maytansinol by Streptomyces); and (7)4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

The cytotoxic conjugates may be prepared by in vitro methods. To link acytotoxic agent, drug or prodrug to the antibody, commonly, a linkinggroup is used. Suitable linking groups are known in the art and includedisulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. For example,conjugates can be constructed using a disulfide exchange reaction or byforming a thioether bond between an antibody of interest and the drug orprodrug.

The molecule conjugated to an antibody of interest can be a moleculewith a pharmacologic activity, such as a drug, such as a small moleculeor a biologic. Thus, the biologic can be a cytokine, for example. Themolecule can be a prodrug, such as a drug ester. The molecule can be aradionuclide.

As discussed above, the instant invention provides isolated nucleic acidsequences encoding an antibody or functional variant thereof asdisclosed herein, vector constructs comprising a nucleotide sequenceencoding the extended Type I glycosphingolipid-binding polypeptides ofthe present invention, host cells comprising such a vector, andrecombinant techniques for the production of the polypeptide that bindsextended Type I glycosphingolipids.

The vector normally contains components known in the art and generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, a promoter, a polyA sequence, one ormore marker or selection genes, sequences facilitating and/or enhancingtranslation, an enhancer element and so on. Thus, the expression vectorsinclude a nucleotide sequence operably linked to such suitabletranscriptional or translational regulatory nucleotide sequences such asthose derived from mammalian, microbial, viral or insect genes. Examplesof additional regulatory sequences include operators, mRNA ribosomalbinding sites, and/or other appropriate sequences which controltranscription and translation, such as initiation and terminationthereof. Nucleotide sequences are “operably linked” when the regulatorysequence functionally relates to the nucleotide sequence for theappropriate polypeptide. Thus, a promoter nucleotide sequence isoperably linked to, e.g., the antibody heavy chain sequence if thepromoter nucleotide sequence controls the transcription of thatnucleotide sequence.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with antibody heavy and/or light chain sequencescan be incorporated into expression vectors. For example, a nucleotidesequence for a signal peptide (secretory leader) may be fused in-frameto the polypeptide sequence so that the antibody is secreted to theperiplasmic space or into the medium. A signal peptide that isfunctional in the intended host cells enhances extracellular secretionof the appropriate antibody or portion thereof. The signal peptide maybe cleaved from the polypeptide on secretion of antibody from the cell.Examples of such secretory signals are well known and include, e.g.,those described in U.S. Pat. Nos. 5,698,435; 5,698,417; and 6,204,023.

The vector may be a plasmid, a single-stranded or double-stranded viralvector, a single-stranded or double-stranded RNA or DNA phage vector, aphagemid, a cosmid or any other carrier of a transgene of interest. Suchvectors may be introduced into cells as polynucleotides by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors also may be introduced into cells aspackaged or encapsulated virus, or a

The antibodies of the present invention can be expressed from anysuitable host cell. Examples of host cells useful in the instantinvention include prokaryotic, yeast or eukaryotic cells and include butare not limited to microorganisms such as bacteria (e.g., E. coli, B.subtilis, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella,Serratia and Shigella, as well as Bacilli, Pseudomonas and Streptomyces)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing the antibody coding sequences ofinterest; yeast (e.g., Saccharomyces, Pichia, Actinomycetes,Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma, Neurospora,and filamentous fungi, such as Neurospora, Penicillium, Tolypocladiumand Aspergillus) transformed with recombinant yeast expression vectorscontaining antibody coding sequences; insect cell systems infected withrecombinant virus expression vectors (e.g., Baculovirus) containingantibody coding sequences; plant cell systems infected with recombinantvirus expression vectors (e.g., cauliflower mosaic virus, CaMV; ortobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression Vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293 or 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;or the vaccinia virus 7.5K promoter).

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids, such as pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec,Madison, Wis.), pET (Novagen, Madison, Wis.) and the pRSET (Invitrogen,Carlsbad, Calif.) series of vectors (Studier, J Mol Biol 219:37 (1991);and Schoepfer, Gene 124:83 (1993)). Promoter sequences commonly used forrecombinant prokaryotic host cell expression vectors include T7(Rosenberg et al., Gene 56:125 (1987)), β-lactamase (penicillinase),lactose promoter (Chang et al., Nature 275:615 (1978); and Goeddel etal., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddelet al., Nucl Acids Res 8:4057 (1980)), and tac promoter (Sambrook etal., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory (1990)).

Yeast vectors will often contain an origin of replication sequence, suchas, from a 2μ yeast plasmid, an autonomously replicating sequence (ARS),a promoter region, sequences for polyadenylation, sequences fortranscription termination and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J BiolChem 255:2073 (1980)) or other glycolytic enzymes (Holland et al.,Biochem 17:4900 (1978)) such as enolase, glyceraldehydes-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Fleer et al., Gene 107:285 (1991).Other suitable promoters and vectors for yeast and yeast transformationprotocols are well known in the art. Yeast transformation protocols arewell known. One such protocol is described by Hinnen et al., Proc NatlAcad Sci 75:1929 (1978), which selects for Trp⁺ transformants in aselective medium.

Any eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples include plant and insect cells (Luckow etal., Bio/Technology 6:47 (1988); Miller et al., Genetic Engineering,Setlow et al., eds., vol. 8, pp. 277-9, Plenum Publishing (1986); andMaeda et al., Nature 315:592 (1985)). For example, Baculovirus systemsmay be used for production of heterologous proteins. In an insectsystem, Autographa californica nuclear polyhedrosis virus (AcNPV) may beused as a vector to express foreign genes. The virus grows in Spodopterafrugiperda cells. The antibody coding sequence may be cloned undercontrol of an AcNPV promoter (for example, the polyhedrin promoter).Other hosts that have been identified include Aedes, Drosophilamelanogaster and Bombyx mori. A variety of viral strains fortransfection are publicly available, e.g., the L-1 variant of AcNPV andthe Bm-5 strain of Bombyx mori NPV. Moreover, plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, algae, duckweed andtobacco can also be utilized as hosts, as known in the art.

Vertebrate cells, and propagation of vertebrate cells, in culture(tissue culture) can be a routine procedure, although fastidious celllines do exist which require, for example, a specialized medium withunique factors, feeder cells and so on, see Tissue Culture, Kruse etal., eds., Academic Press (1973). Examples of useful mammal host celllines are monkey kidney; human embryonic kidney; baby hamster kidney;Chinese hamster ovary (CHO, Urlaub et al., Proc Natl Acad Sci USA77:4216 (1980)); mouse Sertoli; human cervical carcinoma (for example,HeLa); canine kidney; human lung; human liver; mouse mammary tumor; andNSO cells.

Host cells are transformed with vectors for antibody production andcultured in conventional nutrient medium containing growth factors,vitamins, minerals and so on, as well as inducers appropriate for thecells and vectors used. Commonly used promoter sequences and enhancersequences are derived, for example, from polyoma virus, Adenovirus 2,Simian virus 40 (SV40) and human cytomegalovirus (CMV). DNA sequencesderived from the SV40 viral genome may

Commercially available medium such as Ham's F10, Minimal EssentialMedium (MEM), RPMI-1640 and Dulbecco's Modified Eagle's Medium (DIEM)are suitable for culturing host cells. In addition, any of the mediadescribed in Ham et al., Meth Enzymol 58:44 (1979) and Barnes et al.,Anal Biochem 102:255 (1980), and in U.S. Pat. No. 4,767,704; 4,657,866;4,560,655; 5,122,469; 5,712,163; or 6,048,728 may be used as a culturemedium for the host cells. Any of those media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin or epidermal growth factor), salts (such as chlorides, suchas sodium, calcium or magnesium chloride; and phosphates), buffers (suchas HEPES), nucleotides (such as adenosine and thymidine), antibiotics,trace elements (which may be defined as inorganic compounds usuallypresent at final concentrations in the micromolar range) and glucose oran equivalent energy source. Any other necessary supplements may beincluded at appropriate concentrations, as a design choice. The cultureconditions, such as temperature, pH and the like, are as known in theart appropriate for the cell and to enable the desired expression of thetransgene.

The polynucleotides of interest may be obtained, and the nucleotidesequence of the polynucleotides determined, by any method known in theart. For example, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,Bio/Techniques 17:242 (1994)) and then amplifying the ligatedoligonucleotides, for example, by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid of a cell expressing same. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be obtained from a suitable source, such as alibrary, which may be one specific for antibody-producing cells, such ashybridoma cells selected to express an antibody of the invention.Suitable primers can be configured for PCR amplification. Amplifiednucleic acids generated by PCR may then be cloned into replicablecloning vectors using any method known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody are determined, the nucleotide sequence of the antibody maybe manipulated to obtain the equivalents of interest described hereinusing methods known in the art for manipulating nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR etc.(see, for example, Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory (1990); and Ausubel etal., eds., Current Protocols in Molecular Biology, John Wiley & Sons(1998)) to generate antibodies having a different amino acid sequence,for example, to create amino acid substitutions, deletions and/orinsertions.

The amino acid sequence of the heavy and/or light chain variable domainmay be inspected to identify the sequences of the CDR's by well knownmethods, e.g., by comparison to known amino acid sequences of otherheavy and light chain variable regions to determine the regions ofsequence hypervariability. Using routine recombinant DNA techniques, oneor more of the CDR's may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The polynucleotide of interest generated by thecombination of the framework regions and one or more CDR's, encodes amolecule that specifically binds extended Type I glycosphingolipid, orat least the carbohydrate epitopes and structure recognized thereby. Forexample, such methods may be used to make amino acid substitutions ordeletions of one or more cysteine residues participating in anintrachain disulfide bond to generate antibody molecules lacking one ormore intrachain disulfide bonds.

The antibodies or antibody fragments of the invention can be used todetect extended Type I glycosphingolipid, and hence cells expressingextended Type I glycosphingolipid, in a biological sample in vitro or invivo. In one embodiment, the anti-extended Type I glycosphingolipidantibody of the invention is used to determine presence of and the levelof extended Type I glycosphingolipid in a tissue or in cells derivedfrom the tissue. The levels of extended Type I glycosphingolipid in thetissue or biopsy can be determined, for example, in an immunoassay withthe antibodies or antibody fragments of the invention. The tissue orbiopsy thereof can be frozen or fixed. The same or other methods can beused to determine other properties of extended Type I glycosphingolipid,such as the level thereof, cellular localization and so on.

The above-described method can be used, for example, to diagnose acancer in a subject known to have or suspected to have a cancer, whereinthe level of extended Type I glycosphingolipid measured in said patientis compared with that of a normal reference subject or standard.

The instant invention further provides for monoclonal antibodies,humanized antibodies and epitope-binding fragments thereof that arefurther labeled for use in research or diagnostic applications. In someembodiments, the label is a radiolabel, a fluorophore, a chromophore, animaging agent or a metal ion, for example.

A method for diagnosis is also provided in which said labeled antibodiesor epitope-binding fragments thereof are administered to a subjectsuspected of having a cancer, arthritis, autoimmune diseases or otherdiseases related to, caused by or associated with extended Type Iglycosphingolipid expression and/or function, and the distribution ofthe label within the body of the subject is measured or monitored.

The antibody and fragments thereof of the instant invention may be usedas affinity purification agents. In that process, the antibodies areimmobilized on a solid phase, such as a dextran or agarose, resin orfilter paper, using methods known in the art. The immobilized antibodyis contacted with a sample containing extended Type I glycosphingolipidor cells carrying same to be purified, and thereafter the support iswashed with a suitable solvent that will remove substantially all thematerial in the sample except the extended Type I glycosphingolipid orcell to be purified, which is bound to the immobilized antibody ofinterest. Finally, the support is washed with another suitable solvent,such as glycine buffer, pH 5.0, that will release the extended Type Iglycosphingolipid or cell from the antibody of interest.

For diagnostic applications, the antibody of interest typically will belabeled with a detectable moiety or marker. Numerous labels areavailable which can be generally grouped into the following categories:(a) radioisotopes, such as ³⁶S, ¹⁴C, ¹²⁵I, ³H and ¹³¹I (the antibody canbe labeled with the radioisotope using a technique described in, forexample, Current Protocols in Immunology, vol. 12, Coligen et al., ed.,Wiley-Interscience, New York (1991), and radioactivity can be measuredusing scintillation counting); (b) fluorescent labels, such as rareearth chelates (europium chelates), fluorescein and derivatives thereof;rhodamine and derivatives thereof, dansyl, lissamine, phycoerythrin andTexas Red, the fluorescent labels can be conjugated to the antibodyusing a technique disclosed in Current Protocols in Immunology, supra,for example, where fluorescence can be quantified using a fluorimeter;and (c) various enzyme substrate labels are available (U.S. Pat. No.4,275,149 provides a review), the enzyme generally catalyzes a chemicalalteration of a chromogenic substrate which can be measured usingvarious techniques, for example, the enzyme may catalyze a color changein a substrate, which can be measured spectrophotometrically, or theenzyme may alter the fluorescence or chemiluminescence of the substrate.Techniques for quantifying a change in fluorescence are known, forexample, using a luminometer, or the label donates energy to afluorescent acceptor. Examples of enzymatic labels include luciferases(e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.4,737,456), luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase, such as horseradish peroxidase(HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase and thelike. Techniques for conjugating enzymes to antibodies are described inO'Sullivan et al., Meth Enz, ed. Langone & Van Vunakis, Academic Press,New York, 73 (1981).

When such labels are used, suitable substrates are available, such as:(i) for horseradish peroxidase with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB)); (ii) for alkaline phosphatase (AP) withp-nitrophenyl phosphate as the chromogenic substrate; and (iii)β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate such as,4-methylumbelliferyl-β-D-galactosidase.

Other enzyme-substrate combinations are available to those skilled inthe art. For a general review, see U.S. Pat. Nos. 4,275,149 and4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Forexample, the antibody can be conjugated with biotin and any of thereporters mentioned above can be conjugated with avidin, or vice versa.Biotin binds selectively to avidin and thus, the label can be conjugatedwith the antibody in that indirect manner. Various avidins are known inthe art. Alternatively, to achieve indirect conjugation of the label,the antibody can be conjugated with a small hapten (e.g., digoxin) andone of the different types of labels or reporters mentioned above isconjugated with an anti-digoxin antibody. Thus, indirect conjugation ofthe label with the antibody or mutein can be achieved using a secondantibody.

In another embodiment of the invention, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody, another form of a second antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, determinant or epitope, ofthe target to be detected. In a sandwich assay, the test sample to beanalyzed is bound by a first antibody which is immobilized directly orindirectly on a solid support, and thereafter a second antibody directlyor indirectly labeled binds to the bound test sample, thus forming aninsoluble three-part complex, see e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody or other suitable member of the binding pair (antibody/antigen,receptor/ligand, enzyme/substrate, for example) that is labeled with adetectable moiety (indirect sandwich assay). For example, one type ofsandwich assay is an ELISA assay, in which case the detectable moiety isan enzyme.

For immunohistochemistry, the cell or tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin, for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody or variant thereof is labeled with aradionucleotide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so thatthe sites expressing extended Type I glycosphingolipid can be localizedusing, for example, immunoscintigraphy and a gamma camera.

The instant invention also includes kits, e.g., comprising an antibody,fragment thereof, homolog, derivative thereof and so on, such as alabeled or cytotoxic conjugate, and instructions for the use of theantibody, conjugate for killing or labeling particular cell types and soon. The instructions may include directions for using the antibody,conjugate and so on in vitro, in vivo or ex vivo. The antibody can be inliquid form or as a solid, generally lyophilized. The kit can containsuitable other reagents, such as a buffer, a reconstituting solution andother necessary ingredients for the intended use. A packaged combinationof reagents in predetermined amounts with instructions for use thereof,such as, for a therapeutic use for performing a diagnostic assay iscontemplated. Where the antibody is labeled, such as with an enzyme, thekit can include substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied toprovide for concentrates of a solution of a reagent, which provides userflexibility, economy of space, economy of reagents and so on. Thereagents may be provided as dry powders, usually lyophilized, includingexcipients, which on dissolution provide a reagent solution having theappropriate concentration.

The antibodies of the present invention may be used to treat a mammal.In one embodiment, the antibody or equivalent of interest isadministered to a nonhuman mammal for the purposes of obtainingpreclinical data, for example. Exemplary nonhuman mammals to be treatedinclude nonhuman primates, dogs, cats, rodents and other mammals inwhich preclinical studies are performed. Such mammals may be establishedanimal models for a disease to be treated with the antibody, or may beused to study toxicity of the antibody of interest. In each of thoseembodiments, dose escalation studies may be performed in the mammal. Theproduct of interest may have therapeutic use in those animals as well.

An antibody, with or without a second component, such as a therapeuticmoiety conjugated to same, administered alone or in combination with acytotoxic factor(s) can be used as a therapeutic. The present inventionis directed to antibody-based therapies which involve administeringantibodies of the invention to an animal, a mammal or a human, fortreating an extended Type I glycosphingolipid-mediated or associateddisease, disorder or condition. The animal or subject may be a mammal inneed of a particular treatment, such as a mammal having been diagnosedwith a particular disorder, e.g., one relating to extended Type Iglycosphingolipid, or associated with abnormal extended Type I chainstructure expression and function. Antibodies directed against extendedType I glycosphingolipid are useful, for example, for prophylaxis ortreatment of cancer and autoimmune disorders, for example. For example,by administering a therapeutically acceptable dose of an anti-extendedType I glycosphingolipid antibody of the instant invention, or acocktail of a plurality of the instant antibodies or equivalentsthereof, or in combination with other antibodies of varying sources, orin combination with a non-antibody drug, such as, an anti-inflammatorydrug, a cytotoxic agent, an antibiotic and so on, such as, a platinumdrug, methotrexate and so on, disease symptoms may be ameliorated orprevented in the treated mammal, particularly humans.

Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs, equivalentsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention as described herein (including fragments,analogs and derivatives thereof) and anti-idiotypic antibodies asdescribed herein. The antibodies of the invention can be used to treat,inhibit or prevent diseases, disorders or conditions associated withaberrant expression and/or activity of extended Type Iglycosphingolipid, including, but not limited to, any one or more of thediseases, disorders, or conditions described herein. The treatmentand/or prevention of diseases, disorders or conditions associated withaberrant expression and/or activity of extended Type I glycosphingolipidincludes, but is not limited to, alleviating at least one symptomassociated with those diseases, disorders, or conditions. Antibodies ofthe invention may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein. The term“physiologically acceptable,” “pharmacologically acceptable,”“pharmaceutically acceptable,” and so on means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals and more particularly in humans.

The anti-extended Type I glycosphingolipid antibody can be administeredto a mammal in any acceptable manner. Methods of introduction include,but are not limited to, parenteral, subcutaneous, intraperitoneal,intrapulmonary, intranasal, epidural, inhalation and oral routes, and ifdesired for immunosuppressive treatment, intralesional administration.Parenteral infusions include intramuscular, intradermal, intravenous,intraarterial or intraperitoneal administration. The antibodies orcompositions may be administered by any convenient route, for example,by infusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosaetc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the therapeutic antibodies or compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Inaddition, the antibody can be suitably administered by pulse infusion,particularly with declining doses of the antibody. Preferably the dosingis given by injection, preferably intravenous or subcutaneousinjections, depending, in part, on whether the administration is briefor chronic.

Various other delivery systems are known and can be used to administeran antibody of the present invention, including, e.g., encapsulation inliposomes, microparticles, microcapsules and so on (see Langer, Science249:1527 (1990)); expression of an antibody, mutein thereof orantigen-binding portion thereof, of interest on a liposome, particle,capsule and so on to yield a targeting vehicle, Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein et al., eds., p. 353-365 (1989); and Lopez-Berestein,ibid., p. 317-327; recombinant cells capable of expressing the compound,see, e.g., Wu

The active ingredients may also be entrapped in a microcapsule prepared,for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nanoparticles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed. (1980).When the liposome or particle expresses an antibody of interest, any ofa variety of compounds can be carried in the liposome, such as, anon-antibody drug, small molecule drug and so on. The instant antibodycan thus serve a targeting function.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. Theantibody may also be administered into the lungs of a patient in theform of a dry powder composition, see e.g., U.S. Pat. No. 6,514,496.

In a specific embodiment, it may be desirable to administer thetherapeutic antibodies or compositions of the invention locally to thearea in need of treatment; that may be achieved by, for example, and notby way of limitation, local infusion, topical application, by injection,by means of a catheter, by means of a suppository or by means of animplant, said implant being of a porous, non-porous or gelatinousmaterial, including membranes, such as sialastic membranes or fibers.Preferably, when administering an antibody of the invention, care istaken to use materials to which the protein does not absorb or adsorb.

In yet another embodiment, the antibody can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,Science 249:1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N Engl J Med321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer et al., eds.,CRC Press (1974); Controlled Drug Bioavailability, Drug Product Designand Performance, Smolen et al., eds., Wiley (1984); Ranger et al., JMacromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann Neurol 25:351 (1989); andHoward et al., J Neurosurg 71:105 (1989)). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films or matrices. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethylmethacrylate), poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers (such as, injectable microspherescomposed of lactic acid-glycolic acid copolymer) andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. Rational strategies can be devised for stabilization dependingon the mechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, amino acid substitutionand developing specific polymer matrix compositions.

Therapeutic formulations of the polypeptide or antibody may be preparedfor storage as lyophilized formulations or aqueous solutions by mixingthe polypeptide having the desired degree of purity with optional“pharmaceutically acceptable” carriers, diluents, excipients orstabilizers typically employed in the art, i.e., buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants and other miscellaneous additives, see Remington'sPharmaceutical Sciences, 16th ed., Osol, ed. (1980). Such additives aregenerally nontoxic to the recipients at the dosages and concentrationsemployed, hence, the excipients, diluents, carriers and so on arepharmaceutically acceptable.

An “isolated” or “purified” antibody is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourceor medium from which the protein is derived, or substantially free ofchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof an antibody in which the polypeptide/protein is separated fromcellular components of the cells from which same is isolated orrecombinantly produced. Thus, an antibody that is substantially free ofcellular material includes preparations of the antibody having less thanabout 30%, 20%, 10%, 5%, 2.5% or 1%, (by dry weight) of contaminatingprotein or cellular or subcellular material. When the antibody isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, 5%, 2.5% or 1% of the volume of the protein preparation. Whenantibody is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals andreagents, i.e., the antibody of interest is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly, such preparations of the antibody have less thanabout 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors orcompounds other than antibody of interest. In a preferred embodiment ofthe present invention, antibodies are isolated or purified.

As used herein, the phrase “low to undetectable levels of aggregation”refers to samples containing no more than 5%, no more than 4%, no morethan 3%, no more than 2%, no more than 1% and often no more than 0.5%aggregation of antibody or variant thereof, that is, two or moreantibody molecules or variants thereof joined or coalesced together, byweight protein, as measured by, for example, high performance sizeexclusion chromatography (HPSEC).

As used herein, the term “low to undetectable levels of fragmentation”refers to samples containing equal to or more than 80%, 85%, 90%, 95%,98% or 99% of intact antibody molecule or variant thereof, of the totalprotein, for example, in a single peak, as determined by HPSEC, or intwo (2) peaks (heavy chain and light chain) by, for example, reducedcapillary gel electrophoresis (rCGE) and containing no other singlepeaks having more than 5%, more than 4%, more than 3%, more than 2%,more than 1% or more than 0.5% of the total protein, each. The rCGE asused herein refers to capillary gel electrophoresis under reducingconditions sufficient to reduce disulfide bonds in an antibody orantibody-type or derived molecule.

The instant invention provides methods for preparing liquid formulationsof the antibody or extended Type I glycosphingolipid-binding fragmentthereof, said methods comprising concentrating a fraction of purifiedantibody to a final concentration of about 15 mg/ml, about 20 mg/ml,about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 200 mg/ml,about 250 mg/ml, about 300 mg/ml or more using, for example, asemi-permeable membrane with an appropriate molecular weight (mw) cutoff(e.g., 30 kD cutoff for F_((ab′)2) fragments thereof; and 10 kD cutofffor F_(ab) fragments) and, optionally, diafiltering the concentratedantibody fraction into the formulation buffer using the same membrane.

In addition, the present invention also encompasses stable liquidformulations of the products of interest that can have improvedhalf-life in vivo. Thus, the antibody of interest has a half-life in asubject, preferably a human, of greater than 3 days, greater than 7days, greater than 10 days, greater than 15 days, greater than 25 days,greater than 30 days, greater than 35 days, greater than 40 days,greater than 45 days, greater than 2 months, greater than 3 months,greater than 4 months, greater than 5 months or more.

As used herein, the terms “stability” and “stable” in the context of aliquid formulation comprising an extended Type I glycosphingolipidantibody or binding fragment thereof refer to the resistance of theantibody or antigen-binding fragment thereof in the formulation tothermal and chemical unfolding, aggregation, degradation orfragmentation under given manufacture, preparation, transportation

The instant invention encompasses liquid formulations having stabilityat temperatures found in a commercial refrigerator or freezer found inthe office of a physician or laboratory, such as from about −20° C. toabout 5° C., said stability assessed, for example, by high performancesize exclusion chromatography (HPSEC), for storage purposes, such as,for about 60 days, for about 120 days, for about 180 days, for about ayear, for about 2 years or more. The liquid formulations of the presentinvention also exhibit stability, as assessed, for example, by HSPEC, atroom temperatures, for at least a few hours, such as about one hour,about two hours or about three hours prior to use.

The term, “carrier,” refers to a diluent, adjuvant, excipient or vehiclewith which the therapeutic is administered. Such physiological carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a suitablecarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. The compositionscan take the form of solutions, suspensions, emulsions, tablets, pills,capsules, powders, sustained-release formulations, depots and the like.The composition can be formulated as a suppository, with traditionalbinders and carriers such as triglycerides. Oral formulations caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate etc., flavorants, colorants, odorants and so on.Examples of suitable carriers are described in “Remington'sPharmaceutical Sciences,” Martin. Such compositions will contain aneffective amount of the antibody, preferably in purified form, togetherwith a suitable amount of carrier so as to provide the form for properadministration to the patient. As known in the art, the formulation willbe constructed to suit the mode of administration.

Buffering agents help to maintain the pH in a range which approximatesphysiological conditions or conditions conducive to antibody stability.Buffers are preferably present at a concentration ranging from about 2mM to about 50 mM. Suitable buffering agents for use with the instantinvention include both organic and inorganic acids, and salts thereof,such as, for example, citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture etc.), succinate buffers (e.g., succinicacid-monosodium succinate mixture, succinic acid-sodium hydroxidemixture, succinic acid-disodium succinate mixture etc.), tartratebuffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixtureetc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium gluconate mixture etc.), oxalate buffers (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture etc.) and acetate buffers(e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxidemixture etc.). Phosphate buffers, carbonate buffers, histidine buffers,trimethylamine salts such as Tris, HEPES and other such known bufferscan be used.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from about 0.2% to about 1% (w/v). Suitablepreservatives for use with the present invention include phenol, benzylalcohol, m-cresol, methyl paraben, propyl paraben,octadecyldimethylbenzyl ammonium chloride, benzylconium halides (e.g.,chloride, bromide and iodide), hexamethonium chloride, alkyl parabens,such as, methyl or propyl paraben, catechol, resorcinol, cyclohexanoland 3-pentanol.

Isotonicifiers are present to ensure physiological isotonicity of liquidcompositions of the instant invention and include polhydric sugaralcohols, such as, trihydric or higher sugar alcohols, such as glycerin,erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydricalcohols can be present in an amount of between about 0.1% to about 25%,by weight, preferably about 1% to about 5% taking into account therelative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols;amino acids, such as arginine, lysine, glycine, glutamine, asparagine,histidine, alanine, ornithine, L leucine, 2-phenylalanine, glutamicacid, threonine etc.; organic sugars or sugar alcohols, such as lactose,trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol,ribitol, myoinisitol, galactitol, glycerol and the like, includingcyclitols such as inositol; polyethylene glycol; amino acid polymers;sulfur containing reducing agents, such as urea, glutathione, thiocticacid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodiumthiosulfate; low molecular weight polypeptides (i.e., <10 residues);proteins, such as human serum albumin, bovine serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone,saccharides, monosaccharides, such as xylose, mannose, fructose,glucose; disaccharides, such as lactose, maltose and sucrose;trisaccharides such as raffinose; polysaccharides such as dextran and soon. Stabilizers can be present in the range from about 0.1 to about10,000 w/w per part of active protein.

Additional miscellaneous excipients can include bulking agents, (e.g.,agar, gelatin, starch and so on), chelating agents (e.g., EDTA),antioxidants (e.g., ascorbic acid, methionine or vitamin E) andcosolvents.

As used herein, the term “surfactant” refers to organic substanceshaving amphipathic structures, namely, are composed of groups ofopposing solubility tendencies, typically an oil soluble hydrocarbonchain and a water soluble ionic group. Surfactants can be classified,depending on the charge of the surface active moiety, into anionic,cationic and nonionic surfactants. Surfactants often are used aswetting, emulsifying, solubilizing and dispersing agents for variouspharmaceutical compositions and preparations of biological materials asthose discussed herein.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent, as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stresseswithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188etc.), Pluronic® polyols and polyoxyethylene sorbitan monoethers(TWEEN-20®, TWEEN-80® etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

As used herein, the term, “inorganic salt,” refers to any compound,containing no carbon, that results from replacement of part or all ofthe acid hydrogen or an acid by a metal or a group acting like a metal,and often are used as a tonicity adjusting compound in pharmaceuticalcompositions and preparations of biological materials. The most commoninorganic salts are NaCl, KCl, NaH₂PO₄ etc.

The present invention provides liquid formulations of an anti-extendedType I glycosphingolipid-binding compound or fragment thereof, having apH ranging from about 5.0 to about 7.0, or about 5.5 to 6.5, or about5.8 to about 6.2, or about 6.0.

The formulation herein also may contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely impact each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules suitably are present in combination in amountsthat are effective for the purpose intended. The formulation also cancontain another drug, or a small molecule, a pharmacologic agent, suchas an anti-neoplastic drug, such as, cisplatin.

The term “small molecule” and analogous terms include, but are notlimited to, peptides, peptidomimetics, amino acids, amino acidanalogues, organic compounds, pharmacologically active agents, such asdrugs, polynucleotides, polynucleotide analogues, nucleotides,nucleotide analogues, organic or inorganic compounds (i.e., includingheterorganic and/organometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds.

Thus, in the case of cancer, the antibodies of the invention may beadministered alone or in combination with other types of cancertreatments, including conventional chemotherapeutic agents (paclitaxel,carboplatin, cisplatin and doxorubicin), anti-EGFR agents (gefitinib,erlotinib and cetuximab), anti-angiogenesis agents (bevacizumab andsunitinib), as well as immunomodulating agents, such as interferon-α andthalidomide.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment, management oramelioration of a disease, disorder, malady and the like associated withaberrant extended Type I glycosphingolipid expression, and metabolism ingeneral, and activity. Also included are known compounds with apharmacologic effect in treating a disorder and so on that is associatedwith aberrant extended Type I glycosphingolipid expression, metabolismand activity.

The antibody or variant, optionally, is formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages.

The formulations to be used for in vivo administration must be sterile.That can be accomplished, for example, by filtration through sterilefiltration membranes. For example, the liquid formulations of thepresent invention may be sterilized by filtration using a 0.2 μm or a0.22 μm filter.

In addition, the antibodies of the instant invention may be conjugatedto various effector molecules such as heterologous polypeptides, drugs,radionucleotides or toxins, see, e.g., WO 92/08495; WO 91/14438; WO89/12624; U.S. Pat. No. 5,314,995; and EPO 396,387. An antibody orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive metal ion (e.g., a emitters, such as, for example,²¹³Bi). A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, doxorubicin,daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol and puromycin and analogs orhomologues thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil and decarbazine), alkylating agents (e.g.,mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin, daunomycin anddoxorubicin), antibiotics (e.g., dactinomycin, actinomycin, bleomycin,mithramycin and anthramycin (AMC)), and anti-mitotic agents (e.g.,vincristine and vinblastine).

To prolong the serum circulation of an antibody in vivo, varioustechniques can be used. For example, inert polymer molecules, such ashigh molecular weight polyethylene glycol (PEG), can be attached to anantibody with or without a multifunctional linker either throughsite-specific conjugation of the PEG to the N-terminus or to theC-terminus of the antibody or via c amino groups present on lysineresidues. Linear or branched polymer derivatization that results inminimal loss of biological activity can be used. The degree ofconjugation can be closely monitored by SDS-PAGE and mass spectrometryto ensure proper conjugation of PEG molecules to the antibodies.Unreacted PEG can be separated from antibody-PEG conjugates bysize-exclusion or by ion exchange chromatography. PEG-derivatizedantibodies can be tested for binding activity as well as for in vivoefficacy using methods known to those of skilled in the art, forexample, by immunoassays described herein.

An antibody having an increased half-life in vivo can also be generatedby introducing one or more amino acid modifications (i.e.,substitutions, insertions or deletions) into an IgG constant domain, orF_(c)R binding fragment thereof (such as an F_(c) or hinge F_(c) domainfragment), see, e.g., WO 98/23289; WO 97/34631; and U.S. Pat. No.6,277,375.

Further, an antibody can be conjugated to albumin to make an antibodymore stable in vivo or to have a longer half life in vivo. Thetechniques are known in the art, see e.g., WO 93/15199, WO 93/15200 andWO 01/77137; and EPO 413622. The antibody also can be modified, forexample, by glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein and so on.

Techniques for conjugating such a therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., in Monoclonal Antibodies and CancerTherapy, Reisfeld et al. (eds.), Alan R. Liss (1985); Hellstrom et al.,in Controlled Drug Delivery, 2nd ed., Robinson et al., eds., MarcelDekker (1987); Thorpe, in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al., eds. (1985); MonoclonalAntibodies For Cancer Detection and Therapy, Baldwin et al., eds.,Academic Press (1985); and Thorpe et al., Immunol Rev 62:119 (1982).Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate, such as a bifunctional antibody, see,e.g., U.S. Pat. No. 4,676,980.

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (WO 97/33899),AIM II (WO 97/34911), Fas ligand (Takahashi et al., Int Immunol, 6:1567(1994)), VEGF (WO 99/23105); a thrombotic agent; an anti-angiogenicagent, e.g., angiostatin or endostatin; or biological response modifierssuch as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2(IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (GCSF) or othergrowth factors.

The antibody or variant composition will be formulated, dosed andadministered in a manner consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody or variant to beadministered will be governed by such considerations, and can be theminimum amount necessary to prevent, ameliorate or treat an extendedType I glycosphingolipid disease, condition or disorder.

As used herein, the term “effective amount” refers to the amount of atherapy (e.g., a prophylactic or therapeutic agent), which is sufficientto reduce the severity and/or duration of an extended Type Iglycosphingolipid related or associated disease, ameliorate one or moresymptoms thereof, prevent the advancement of an extended Type Iglycosphingolipid related or associated disease or cause regression ofan extended Type I glycosphingolipid related or associated disease, orwhich is sufficient to result in the prevention of the development,recurrence, onset, or progresion of an extended Type I glycosphingolipidrelated or associated disease or one or more symptoms thereof, orenhance or improve the prophylactic and/or therapeutic effect(s) ofanother therapy (e.g., another therapeutic agent) useful for treating anextended Type I glycosphingolipid disease related or associated. Forexample, a treatment of interest can reduce a symptom, based on baselineor a normal level, by at least about 5%, preferably at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100%. In one other embodiment, aneffective amount of a therapeutic or a prophylactic agent reduces asymptom of an extended Type I glycosphingolipid related or associateddisease, such as a cancer, by at least about 5%, preferably at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 100%. Also used herein asan equivalent is the term, “therapeutically effective amount.”

The amount of therapeutic polypeptide, antibody or fragment thereofwhich will be effective in the use or treatment of a particular disorderor condition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Where possible, adose-response curve and the pharmaceutical compositions of the inventioncan be first derived in vitro. If a suitable animal model system isavailable, again a dose-response curve can be obtained and used toextrapolate a suitable human dose practicing methods known in the art.However, based on common knowledge of the art, a pharmaceuticalcomposition effective in promoting a diminution of an inflammatoryeffect, for example, may provide a local therapeutic agent concentrationof between about 5 and about 20 ng/ml, and, preferably, between about 10and about 20 ng/ml

In a preferred embodiment, an aqueous solution of therapeuticpolypeptide, antibody or fragment thereof can be administered bysubcutaneous injection. Each dose may range from about 0.5 mg to about50 mg per kilogram of body weight, or more preferably, from about 3 mgto about 30 mg per kilogram body weight. The dosage can be ascertainedempirically for the particular disease, patient population, mode ofadministration and so on, practicing pharmaceutic methods known in theart.

The dosing schedule for subcutaneous administration may vary from once aweek to daily to multiple times a day depending on a number of clinicalfactors, including the type of disease, severity of disease and thesensitivity of the subject to the therapeutic agent.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to humans. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine or other “caine”anesthetic to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or a concentratein a sealed container, such as an ampoule or sachet indicating thequantity of active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided, for example, in a kit, so that the ingredientsmay be mixed prior to administration.

The invention also provides that a liquid formulation of the presentinvention is packaged in a sealed container such as an ampule or sachetindicating the quantity of the product of interest. The liquidformulations of the instant invention can be in a sealed containerindicating the quantity and concentration of the antibody or antibodyfragment. The liquid formulation of the instant invention can besupplied in a sealed container with at least about 15 mg/ml, 20 mg/ml,30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml,100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml of extendedType I glycosphingolipid antibody in a quantity of about 1 ml, 2 ml, 3ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml or 20 ml, forexample.

An article of manufacture containing materials useful for the treatmentof the disorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for diagnosing,preventing or treating an extended Type I glycosphingolipid condition ordisease and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The label on or associated with thecontainer indicates that the composition is used for treating thecondition of choice. The article of manufacture may further comprise asecond container comprising a pharmaceutically acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including buffers, diluents, filters, needles, syringesand package inserts with instructions for use.

In another aspect of the invention, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of extended Type Iglycosphingolipid, by way of gene therapy. Gene therapy refers totherapy performed by the administration to a subject of an expressed orexpressible nucleic acid of interest. Alternatively, cells manipulatedto carry gene sequences of interest are administered to a host. In anembodiment of the invention, the nucleic acids produce the encodedprotein in and by target host cells that mediate a therapeutic effect.Any of the methods for gene therapy available can be used according tothe instant invention.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488 (1993); Wu et al., Biotherapy 3:87 (1991);Tolstoshev, Ann Rev Pharmacol Toxicol 32:573 (1993); Mulligan, Science260:926 (1993); Morgan et al., Ann Rev Biochem 62:191 (1993); and May,TIBTECH 11:155 (1993).

In one aspect, the compound comprises nucleic acid sequences encoding anantibody, or functional binding fragments thereof, said nucleic acidsequences being part of expression vectors that express the antibody orfragments or chimeric proteins or heavy or light chains thereof in asuitable host. In particular, such nucleic acid sequences have promotersoperably linked to the antibody or antigen-binding coding region, saidpromoter being inducible or constitutive, and, optionally,tissue-specific, as well as other regulatory sequences.

In another particular embodiment, nucleic acid molecules are used inwhich the antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for integration and intrachromosonialexpression of the antibody-encoding nucleic acids (Koller et al., ProcNatl Acad Sci USA 86:8932 (1989); Zijlstra et al., Nature 342:435(1989)). In specific embodiments, the expressed antibody molecule is asingle chain antibody; alternatively, the nucleic acid sequences includesequences encoding both the heavy and light chains, or fragmentsthereof, of the antibody. Alternative methods for integration includeusing particular transcription factors that recognize specific nucleicacid sequences, zinc fingers and so on.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient.

in one embodiment, the nucleic acid sequences are directly administeredin vivo and are expressed to produce the encoded product. That can beaccomplished by any of numerous methods known in the art, e.g., byconstructing the antibody encoding sequences as part of an appropriatenucleic acid expression vector and administering same so that thevectors become intracellular, e.g., by infection using defective orattenuated retroviral or other viral vectors (see U.S. Pat. No.4,980,286), by direct injection of naked DNA, by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), using non-viralvectors, such as synthetic compositions comprising an amphipathiccompound that binds the hydrophilic nucleic acid and has the ability tofuse with cells, generally thus containing a hydrophobic portion forcombining with membranes, coating with lipids or cell-surface receptorsor transfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, by administering the vector in linkage with a peptidewhich is known to enter the nucleus, by administering the vector inlinkage with a ligand subject to receptor-mediated endocytosis (see,e.g., Wu et al., J Biol Chem 262:4429 (1987)) (which can be used totarget cell types specifically expressing the receptors) etc. In anotherembodiment, nucleic acid-ligand complexes can be formed in which theligand comprises a fusogenic viral peptide to disrupt endosomes,allowing the nucleic acid to avoid lysosomal degradation. In yet anotherembodiment, the nucleic acid can be targeted in vivo for cell-specificuptake and expression, by targeting a specific receptor (see, e.g., WO92/06180; WO 92/22635; WO92/20316; WO93/14188 and WO 93/20221).

Regarding vectors, for example, a lentiviral vector can be used as knownin the art. The lentiviral vectors contain components for packaging theviral genome and integration into the host cell DNA. The nucleic acidsequences encoding the antibody to be used in gene therapy are clonedinto one or more vectors, which facilitate the delivery of the gene intoa patient. For example, a lentiviral vector can be used to deliver atransgene to hematopoietic stem cells. References illustrating the useof retroviral vectors in gene therapy are: Clowes et al., J Clin Invest93:644 (1994); Kiem et al., Blood 83:1467 (1994); Salmons et al., HumanGene Therapy 4:129 (1993); and Grossman et al., Curr Opin Gen and Dev3:110 (1993).

Adenoviruses also may be used in the instant invention. Targets foradenovirus-based delivery systems include liver, the central nervoussystem, endothelial cells and muscle, for example. Adenoviruses infectnon-dividing cells, an advantage over early retroviral vectors. Kozarskyet al., Curr Opin Gen Dev 3:499 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431 (1991); Rosenfeld et al., Cell 68:143 (1992);Mastrangeli et al., J Clin Invest 91:225 (1993); WO94/12649; and Wang etal., Gene Therapy 2:775 (1995).

Adeno-associated virus (AAV) also can be used in gene therapy (Walsh etal., Proc Soc Exp Biol Med 204:289 (1993); and U.S. Pat. Nos. 5,436,146;6,632,670; and 6,642,051).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by methods such as electroporation, lipofection,calcium phosphate-mediated transfection or viral infection. Usually, themethod of transfer includes the transfer of a selectable marker to thecells. The cells then are placed under selection to isolate those cellsthat have taken up and are expressing the transferred gene. Those cellsthen are delivered to a patient.

Thus, the nucleic acid can be introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (see, e.g., Loeffler et al., Meth Enzymol 217:599(1993); Cohen et al., Meth Enzymol 217:618 (1993); and Cline, Pharm Ther29:69 (1985)) and may be used in accordance with the present invention,provided that the necessary developmental and physiological functions ofthe recipient cells are not disrupted. The technique should provide forthe stable transfer of the nucleic acid to the cell, so that the nucleicacid is expressed by the cell, heritable and expressed by the cellprogeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously, forexample, as known in the bone marrow transplantation art. The amount ofcells envisioned for use depends on the desired effect, patient stateetc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include, but arenot limited to, epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes, blood cells, such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes and granulocytes, various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver etc.

In one embodiment, the cell used for gene therapy is autologous to thepatient. Nucleic acid sequences encoding an antibody of the instantinvention are introduced into the cells such that the transgene isexpressed by the cells or their progeny, and the recombinant cells thenare administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with the embodiment of the instantinvention (see e.g., WO 94/08598; Stemple et al., Cell 71:973 (1992);Rheinwald Meth Cell Bio 21A:229 (1980); and Pittelkow et al., MayoClinic Proc 61:771 (1986)). Because extended Type I glycosphingolipid isexpressed on, for example, B cells, blood cells and bone marrow cellsare suitable host cells. However, the scope of the instant inventionregarding the use of stem cell hosts does not contemplate the making andusing of a transgene to make a transgenic organism by administering thetransgene of interest to embryos and/or embryonic stem cells.

The invention thus provides methods of treatment, prophylaxis andamelioration of extended Type I glycosphingolipid related and associateddiseases or one or more symptoms thereof by administering to a subjectan effective amount of, for example, a liquid formulation, an antibodyor variant thereof of the invention. The subject is preferably a mammalsuch as non-primate (e.g., cows, pigs, horses, cats, clogs, rats etc.)and a primate (e.g., monkey, such as a cynomolgus monkey, and a human).In a preferred embodiment, the subject is a human.

Extended Type I glycosphingolipid also is expressed on certain cancercells, such as pancreas, colon and bladder, as well as on T cellleukemias (Qinping et al., Oncogene 24:573-584, 2005), and stimulationof extended Type I glycosphingolipid correlated with proliferation ofcarcinoma cells, Meijer et al., Canc Res 66:9576-9582, 2006.

Thus, the antibody or derivative thereof of interest can be used tocontrol proliferation of cancer cells expressing extended Type Iglycosphingolipid, which cancers are identified by determining presenceof extended Type I glycosphingolipid expression by a diagnostic assaytaught herein. The antibody of interest can reduce infiltration ofmalignant cells, reduce resistance to apoptosis and minimizeproliferation. Such patients then are administered a cancer cellproliferation inhibiting amount of an antibody, or derivative thereof,of interest as provided herein. As taught herein, an antibody or antigenbinding portion thereof can be administered to a patient in a number ofways, including administering a polypeptide, a polynucleotide and so on.Essentially any cancer that expresses a Type I epitope of interest canbe detected and/or treated with an antibody of interest. For example,the malignant cell can be an epithelial cell. The epithelial cell can befound in any malignant cell of any organ or tissue origin, such as,colon, rectum, esophagus, lung, prostate, breast, pancreas, the oralcavity, vagina, the gastrointestinal tract in general, urinary tract andso on. However, the cancer need not be limited to an epithelial cell, solong as the malignant cell expresses a Type I epitope of interest.

The invention now will be exemplified for the benefit of the artisan bythe following non-limiting examples that depict some of the embodimentsby and in which the instant invention can be practiced.

EXAMPLES Example 1 Generation of Immunogen

Colo205 cells (ATCC) (Semple et al., Cancer Res 38: 1345-1355, 1978) aregrown in RPMI 1640 medium containing 10% fetal calf serum. Harvestedcells are washed twice with PBS and stored at −20° C. until needed. Cellpellets are extracted with isopropanol-hexane-water (IHW) (55:25:20)followed by Folch partition, DEAE Sephadex chromatography and HPLC on anIatrobead 6RS-8010 column. Gradient elution of the upper-phase neutralfraction is performed in IHW from 55:40:5 to 55:25:20 over 200 minutes.Fractions are collected and pooled according to HPTLC migration inchloroform-methanol-water (50:40:10). The extended Type I chainglycosphingolipids are purified further by preparative TLC on MerckHPTLC plates (Silica Gel 60, Merck, Darmstadt, Germany), see U.S. Pat.No. 6,083,929.

A positive band (by immunostaining with mAb IMH2) which migrated justbelow dimeric Le^(a) antigen is purified as taught herein.

The colorectal adenocarcinoma cells Colo205 (ATCC CCL-222) and DLD-1(ATCC CCL-221) are cultured in RPMI 1640 medium (Invitrogen Co., Cat.No. 31800) supplemented with 1 mM sodium pyruvate (Invitrogen Co., Cat.No. 11360). Other colorectal adenocarcinoma cells, SW1116 (ATCC CCL-233)and HT-29 (ATCC HTB-38), and lung-derived T84 cells (ATCC, CCL-248) areseparately maintained in Leibovitz's L-15 medium (Invitrogen Co., Cat.No. 41300), McCoy's 5a medium (Invitrogen Co., Cat. No. 12330) andDMEM/F12 medium (Invitrogen Co., Cat. No. 12400). The KATO III gastriccarcinoma cells (ATCC HTB-103) are cultured in IMDM medium (InvitrogenCo., Cat. No. 12200). All media used in the studies are supplementedwith 10% fetal calf serum.

Example 2 Generation of Anti-Extended Type I Glycosphingolipid Mabs

KM mice (Kirin Brewery Co., Ltd.) are generated by cross breeding doubletranschromosomic mice and transgenic mice. KM mice possess humanchromosome fragments containing the entire human immunoglobulin heavychain loci and a YAC transgene for half of the human immunoglobulinkappa light chain loci. KM mice are engineered to express neitherendogenous immunoglobulin heavy chain nor kappa light chain. All of theanimals are maintained and handled according to the rules andregulations accepted in the art.

Colo205 cells are injected intraperitoneally into KM mice every 3 weeks(5×10⁶ cells/injection) for a total of 4 injections, followed byinjection of extended Type I chain glycosphingolipids which are isolatedfrom Colo205 cells and adsorbed on lipopolysaccharide (Sigma, L-7011)(Young et al., J. Exp. Med. 150:1008-1019, 1979) every week for 8injections. The anti-Colo205 neutral glycosphingolipid titers ofimmunized mice are monitored by ELISA, using anti-human kappa-HRP(Southern Biotechnology Associates, Cat. No. 9220-05) as the secondaryantibody until the titer reached 1:6000. Three days after the finalinjection, splenocytes from the boosted mouse are fused withP3/NS1/1-Ag4-1 (NS-1) mouse myeloma cells (ATCC TIB-18) practicingmethods known in the art. Hybridomas are screened by ELISA using 96-wellELISA plates (Corstar, Cat. No. 2592) coated with Colo205 neutralglycolipid. Mouse anti-human IgG antibodies conjugated with HRP are usedas the secondary antibody (Southern Biotechnology Associates, Cat. No.9040-05) and teramethylbenzidene (TMB) (Kem-Zn-Tec Diagnostics, Cat. No.4390) is used as the substrate. Hybridoma supernatants showing highreactivity with Colo205 neutral glycolipids are further confirmed byHPTLC immunostaining and by flow cytometry. Clones strongly staining theextended Type I chain glycolipids and showing high binding on thesurface of Colo205 cells are repeatedly subcloned by limiting dilutionuntil stable clones are established. One stable clone is GNX-8.

Example 3 GNX-8 Antibody

Monoclonal antibody is purified from culture supernatants using proteinA Sepharose (GE Healthcare 17-129-79-02) with pH gradient elutionaccording to the manufacturer's suggested procedures. Each fraction iscollected and the presence of antibody is examined by ELISA. Fractionswith Colo205 neutral glycolipid binding activity are pooled and dialyzedagainst PBS (pH 7.4). Purified antibodies are aliquot and stored at −20°C.

The concentration of monoclonal antibody is determined with the Bio-RadProtein Assay kit (Bio-Rad, Cat. No. 500-0006) using IgG as the standardaccording to the manufacturer's recommended procedures.

The isotype of GNX-8 is determined using an ELISA. GNX-8 is a human IgG1and the light chain is kappa.

Purified GNX-8 is applied to 10% SDS-polyacrylamide gels after beingboiled in 2×SDS gel-loading buffer with (reducing condition) or without(non-reducing condition) β-mercaptoethanol. Electrophoresis is conductedusing the Minutesi-PROTEAN3 Electrophoresis System (BIO-RAD) accordingto the manufacturer's recommendations.

GNX-8 separated on reducing SDS-PAGE gels is transferred ontonitrocellulose (NC) membranes (Amersham) and blocked with 3% skim milkin PBS. The membrane is incubated with secondary antibody for 1 hour atroom temperature. HRP-labeled goat anti-human IgG(γ) antibody (Zymed,62-8420) at 1:5000 dilution and HRP-labeled rabbit anti-human kappachain IgG antibody (DAKO, P0129) at 1:2000 dilution are separately usedto detect the heavy chain and light chain of GNX-8. Western Lightning™Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Cat. No.NEL105) is used to develop the signal on BioMax Light Film (KODAK, Cat.No. 1788207).

Under reducing conditions, the molecular weight of the GNX-8 light chainand heavy chain are as expected for an IgG. GNX-8 is a human monoclonalantibody by Western blot with goat anti-human IgG(γ)-HRP and rabbitanti-human kappa chain-HRP as secondary antibodies, separately. GNX-8 isa human antibody by ELISA isotyping.

The pI analysis of GNX-8 is determined by the PhastSystem (Pharmacia).Briefly, an antibody sample and pI standard are applied on an IEFPhastGel 3-9 using a PhastGel Sample applicator 8/1 comb and areseparated according to the manufacturer's protocol. The gel subsequentlyis silver stained in the PhastSystem Development Unit (Pharmacia)according to the manufacturer's protocol.

The pI analysis reveals multiple bands ranging from pH 8.15 to 8.65indicating the possibility of post-translational modifications of theantibody. The high pI indicates that GNX-8 will be soluble atphysiologic pH.

For detecting cell binding activity, 2×10⁵ cells are washed with PBS andincubated with various concentrations of antibody for 30 minutes at roomtemperature. After a PBS wash, FITC-labeled goat anti-human IgG (Fc)antibodies at 1:3000 dilution (ICN, Cat no. 55198) are added to eachcell sample for an additional 30 minutes at room temperature. For CDCstudies, cells after antibody treatment are washed three times with PBSand incubated with 1 μl of propidium iodide solution (Sigma-Aldrich,P4846) for 30 minutes. After a final PBS washing, cells are analyzed ona flow cytometer (BD, FACSort). The results are processed with CELLQuest3.3 (BD).

Example 4 Cytotoxicity Assay

Human colon cancer cell lines, SW1116, Colo205 and DLD-1, are seeded in48-well plates (Corning Costar) at a density of 2×10⁴ cells/well. Afterbeing cultured overnight, the cells are incubated in 500 μl of mediumwith 25% non-inactivated human serum at various antibody concentrationsfor 2 hr. After a PBS wash, the remaining live cells are quantified bypropidium iodide (PI) solution (Sigma-Aldrich, P4846) staining and areanalyzed by flow cytometry. Normal human IgGs purified from normal humanserum are used as a negative control.

In an alternative assay, target cells are labeled by incubation withabout 100 μl of ⁵¹Cr for about 90 min. at about 37° C. After washing(3×) and incubation (about 1 hr at 37° C.), cells (about 1×10⁶ ml) aresuspended in RPMI-1640 supplemented with about 25 mM HEPES buffer andabout 3% bovine serum albumin. About 20 μl of labeled cells, about 100μl of mAb and 25% heat inactivated human serum are mixed in the wells ofmicrotiter U bottom plates (Corning, N.Y.). Non-specific mouse Ig(Sigma, St. Louis, Mo.) can be used as a negative control. After about 4hr incubation, the plates are centrifuged (500×g, 2 min) with a hangingplate holder assembled in a centrifuge, and radioactivity in about 100μl supernatant in each well is measured with a gamma counter. Eachexperimental group can be tested in triplicate. Percent specific lysiscan be calculated according to the formula ([A−B]×100)/C, where A=cpm inlysed experimental cells; B=cpm in unlysed target cells; and C=cpm intotal target cells. Spontaneous release preferably should not exceed 15%of maximally releasable labeled radioactivity.

ADCC assays are performed by the lactate dehydrogenase (LDH) releaseassay (Promega, CytoTox 96® Non-Radioactive Cytotoxicity Assay) usinghuman peripheral blood mononuclear cells (PBMC) as effector cellsprepared from healthy donors using Ficoll-Paque (GE, 71-7167-00). Theassay quantitatively measures lactate dehydrogenase (LDH), a stablecytosolic enzyme that is released on cell lysis. Released LDH in culturesupernatants is measured with a 30 minute coupled enzymatic assay, whichresults in the conversion of a tetrazolium salt (INT) into a redformazan product. The amount of color formed is proportional to thenumber of lysed cells.

Colo205 cells used as target cells are distributed into 96-well U bottomplates (2×10⁴ cells/well) and are incubated with antibodies in thepresence of the PBMC with various E/T ratios for 4 hours at 37° C. TheLDH activity in the supernatant was measured by CytoTox 96®Non-Radioactive Cytotoxicity Assay. The percent specific cytolysis iscalculated according to the following formula: % specificlysis=100×(E−S_(E)−S_(T))/(M−S_(T)) where E is the experimental release(activity in the supernatant from target cells incubated with antibodyand effector cells), S_(E) is the spontaneous release in the presence ofeffector cells (activity in the supernatant from effector cells withmedium alone), S_(T) is the spontaneous release of target cells(activity in the supernatant from target cells incubated with mediumalone), and M is the maximum release of target cells (activity releasedfrom target cells lysed with 9% Triton X-100).

The in vitro antitumor activity of GNX-8 is evaluated by CDC assay.Treatment of human colorectal cancer cells, SW1116, Colo205 and DLD-1,with GNX-8 in the presence of 25% human serum, results in substantialcell lysis in a dose dependent manner. The results indicate that GNX-8kills target cells through complement-dependent cytolysis.

The CDC effect on SW1116 and Colo205 cells in some experiments isstronger than that on DLD-1 cells. The viability of cells is inverselyproportional proportion to the level of expression of GNX-8 antigen. TheCDC effect of GNX-8 and levels of GNX-8 antigen expression on the threecolorectal cancer lines demonstrate that cancer cells with higher GNX-8antigen expression are more susceptible to cytotoxicity while those withlower GNX-8 antigen expression have higher viability. The results leadto the conclusion that the antitumor activity of GNX-8 can depend on theexpression level of GNX-8 antigen. Patients with high GNX-8 antigenexpression on tumor cells might be treated with GNX-8 alone, whiletumors expressing lower levels of GNX-8 antigen, may benefit from acombination therapy with one or more other cancer drugs in addition toGNX-8 antibody.

ADCC activity of human peripheral blood mononuclear cells (PBMC) isevaluated against human colorectal cancer Colo205 in the presence ofGNX-8. ADCC activity with IMH2 is used as the positive control and humanIgG as the negative control.

GNX-8 induces strong ADCC activity against Colo205 cells. The cytotoxiceffect is correlated positively with both E/T ratio and GNX-8concentration. One hundred percent cell lysis is observed at E/T ofabout 20/1. A maximal ADCC effect, and a trend observed for about 50%lysis as well, is observed at about 5 μg/ml of GNX-8 and at about 50μg/ml for IMH2, respectively. Control human IgG shows no cytotoxiceffect regardless of E/T ratio or IgG concentration. The dose of GNX-8to reach 50% lysis is less than 1/10 needed for IMH2.

Example 5 Biacore Affinity Analysis

Type I glycosphingolipids are affixed to a chip. Then the mAb's areexposed to the chip for kinetic measurements and epitope sequenceanalysis surrounding the antibody-antigen binding reaction, followingthe manufacturer's recommendations (GE Healthcare, Pistcataway, N.J.).

Example 6 In Vivo Assay

Antitumor activity of GNX-8 is evaluated in a Colo205 xenograft model.Colo205 cells are washed twice with PBS and reconstituted at a celldensity of 5×10⁶/100 μl in PBS. Female nude mice of age 6-8 weeks areinoculated s.c. with 100 μl of the Colo205 cell suspension in the flankregion. Tumor sizes are measured three times a week with a verniercaliper and tumor weights (mg) are estimated as (width²×length)/2. GNX-8or normal human IgG is i.p. injected in tumor-bearing nude miceaccording to designed doses and schedules.

To evaluate the in vivo antitumor efficacy of GNX-8, cancer cells (5×10⁶cells/mouse) are injected in nude mice and treated with either GNX-8(treatment group; 8 mice/group) or normal human IgG (control group, 7mice/group) 24 hours after tumor inoculation. Five doses (300 μg/mouse)at 24-hour intervals and subsequently four doses (600 μg/mouse) at48-hour intervals are injected in both groups.

Tumor growth is significantly inhibited in GNX-8-treated mice. Thetreatment group reaches a median tumor weight of T/C (Treatment/Control)of about 23% on day 11 and continues at that approximate level to theend of the study. A T/C measure of <42% is considered significant indemonstrating antitumor activity.

Half ( 4/8) of the mice in the GNX-8 treatment group achieve long termtumor-free survival over 50 days. On the other hand, tumor size of thecontrol group animals continually increases during the study.

A similar study is conducted in a Colo205 xenograft nude mice model. Thefirst dose of GNX-8 is given at a tumor size of 80 to 100 mg. GNX-8(treatment group) and normal human IgG (control group) are injected once(300 μg/mouse) daily for five days, and with two similar doses at days17 and 21.

Significant tumor inhibition also is observed in the treatment group,although the treatments are discontinued after only 5 doses. The mediantumor weight of T/C (Treatment/Control) is lower than 42% after day 10and through to the end of the study.

To determine whether host effector function contributed to GNX-8efficacy, SCID mice bearing Colo205 xenografts are treated with GNX-8 ornormal human IgG at 600 μg/mouse twice weekly for three weeks. Tumorsize is measured twice every week until the tumor size reached 10% ofthe body weight, which is considered the endpoint of the study.

Survivability is prolonged in the treatment group.

To explore the occurrence of the GNX-8 epitope on human colorectalcancers, several human colorectal cancer cell lines are analyzed.

For example, the in vivo inhibition of DLD-1 by GNX-8 is significant.

Example 7 GNX-8 Antigen

For the analysis of cell glycoprotein, cultured cells are scraped fromthe T-75 flasks and washed twice with PBS, followed by lysis buffer (50mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate,0.1% SDS and 1 mM PMSF). The lysates are passed through a 26 gaugeneedle several times to disperse any large aggregates. Proteinconcentration is determined by Protein Assay Kit (Bio-Rad). The lysatescontaining the same amount of proteins are separated on a gel andanalyzed by Western blot with GNX-8 as the primary antibody and mouseanti-human IgG (Fc) labeled with HRP (Southern Biotechnology Associates,#9040-05) as the secondary antibody. Western Lightning™Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Cat. No.NEL105) is used to develop the signal on BioMax Light Film (KODAK, Cat.No. 1788207).

Neutral glycolipids (2 μl/sample) are spotted on a HPTLC plate (Merck,1.05642, silica gel 60 F₂₅₄), and developed with a mobile phasecontaining chloroform:methanol:water at a ratio of 50:40:10 (V:V:V). Forglycan staining of glycolipids, 0.2% orcinol (Sigma, 0-1875) in 10%H₂SO₄ is sprayed onto a HPTLC plate and incubated for 10 minutes in a110° C. in an oven. For immunostaining, the HPTLC plate is first fixedwith 0.5% poly(isobutyl methacrylate) (Aldrich, 181544) inchloroform:hexane, 1:9 (V:V) for 45 seconds, followed by blocking for 10minutes in 3% BSA/PBS. The plates then are washed with PBS and incubatedwith the primary antibody at room temperature for 1 hr, followed bybiotinylated secondary antibody at room temperature for 1 hr. AnAvidin-Biotin Complex kit (Vector Laboratories, Burlingame, Calif.) isused to amplify signals from the secondary antibody. The plate isincubated at room temperature for 30 minutes, followed by colordevelopment with an Immunostaining HRP-1000 kit (Konica Minolta, 130990)according to the manufacturer's protocol.

To a lyophilized sample, 20 μl of 48% hydrogen fluoride (HF) (Merck) areadded, and then the mixture is incubated at 4° C. for 48 h. At the endof the reaction, HF is removed with N₂ gas. The defucosylatedglycolipids are used for specificity study of GNX-8.

The neutral glycolipids of Colo205 are treated with HF and analyzed byMALDI-TOF MS to confirm the removal of fucose. TLC immunostaining isperformed to analyze the specificity of GNX-8 directed to Colo205neutral glycolipids before and after HF treatment.

GNX-8 recognizes uncleaved glycolipids but not the defucosylated forms,suggesting that the epitope of GNX-8 is a carbohydrate moiety and fucoseis an essential component of the structure.

The specificity of GNX-8 is characterized further by HPTLCimmunostaining on neutral and monosialyl glycolipids isolated fromColo205 cells. One hundred grams of Colo205 cells are collected, andglycolipid fractions are extracted. Colo205 glycolipids separated by TLCare stained for carbohydrate with orcinal/H₂SO₄. The positions ofLe^(a), Le^(b), Le^(a)-Le^(a) and Le^(b)-Le^(a) are identified accordingto Stroud et al. (1992) supra. Sialyl Le^(a) (SLe^(a)) is indicated bystaining with mAb NKH3 (U.S. Pat. No. 5,240,833) and was lateridentified with MALDI-MS. HPTLC immunostaining of the same glycolipidfractions are conducted with CF4C4 (U.S. Pat. No. 5,011,920)(anti-Le^(a)), T218 (Abeam, Cambridge, Mass.) (anti-Le^(b)), IMH2(Stroud et al., 1992, supra) (anti-Le^(b)-Le^(a)), and GNX-8 antibodies.

The results indicate that GNX-8 strongly reacts with extended Type Ichain glycolipids. GNX-8 did not bind to Le^(a) extended Type I chains.The monosialyl glycolipids of Colo205 cells are not recognized by GNX-8.GNX-8 shows very slight cross-reactivity with Le^(b) at higherconcentration (0.6 μg/ml). Unlike IMH2, GNX-8 did not bind to Le^(x) orto Le^(y). GNX-8 binds to an extended chain containing Le^(b). GNX-8binds to Le^(b)-Le^(a).

In addition to TLC immunostaining, the epitope of GNX-8 is characterizedby competitive ELISA using synthetic glycans Le^(b), Le^(a)-Le^(x),Le^(b)-Le^(x) and Le^(x)-Le^(x) as inhibitors, and theLe^(b)-Le^(a)/Le^(a)-Le^(a) glycolipid mixture as a positive control.

The results indicate that GNX-8 slightly cross-reacts with Le^(b)-Le^(x)at high inhibitor concentration, but has no reactivity with other testedsynthetic glycans including synthetic Le^(b) glycans. The bindingactivity of GNX-8 to extended Le^(b) is 1000 times higher than that tosimple Le^(b).

Based on the results, the epitope of GNX-8 likely is an Le^(b) structureon an extended Type I chain with fucosylation, but it is not a simpleLe^(b).

Example 8 Cell and Tissue Distribution

Formalin-fixed paraffin-embedded specimens of human normal and cancertissues are obtained, for example, from US Biomax.

The formalin-fixed, paraffin-embedded tissue arrays of normal andmalignant human tissues are blocked with 0.1% skim milk in PBS for 30minutes. After an additional 10 minutes incubation with 3% H₂O₂, thetissue arrays are washed thrice with PBS before samples are incubatedwith 0.1% BSA/PBS-diluted biotinylated GNX-8 for 1 hour. Then the tissuesamples are reacted with biotin-streptavidin-peroxidase complex (ABCkit, Vector, #PK-6100) for 30 minutes for signal amplification. The DABPLUS Substrate Kit (Zymed #00-2020) is used to visualize immunoreactivestaining according to the manufacturer's protocol. Counterstaining isperformed using hematoxylin. The results are determined by visualizationunder a light microscope.

The expression of GNX-8 antigen on human cancer cells is evaluated byflow cytometry. A number of human colorectal and gastric tumor celllines, such as, Colo205, HT-29, DLD-1, SW1116, T84 and KATO III, areseparately examined for the expression of GNX-8 antigen by flowcytometry.

Flow cytometric analyses demonstrate that GNX-8 exhibits bindingactivity to all tested cancer cell lines. However, the binding issignificantly stronger to SW1116, Colo205 and DLD-1 cells than to theother tested human cancer cell lines.

In addition, GNX-8 antigen expression is tested on HL60 (a promyelocyticcell line), MCF-7 (a breast cancer cell line) and PANC-1 (a pancreascancer cell line), as well as on a mouse colon cancer cell line, CT26.GNX-8 does not bind to those four cell lines.

Two colorectal cancer cell lines, Colo205 and SW1116, are analyzed byWestern blot. The two cell lines demonstrated strong binding with GNX-8in flow cytometry analyses.

The results show presence of GNX-8 antigen on glycoprotein of Colo205and SW1116 over a molecular weight range from 32 to >175 kDa.Accordingly, GNX-8 antigens are not only in glycolipids but also inglycoproteins.

A variety of specimens from various organs, including both normaltissues and cancer tissues, are separately stained with GNX-8. Stainingpatterns in tissue specimens are evaluated by staining intensity andfrequency of positive cells. Staining is graded on a scale of 1+(10-20%), 2+ (20-50%) or 3+ (>50%), whereas frequency is classifiedbased on the percentage of positive cells in each section.

A strong correlation is observed between GNX-8 antigen expression inprimary and metastatic colorectal carcinomas. An immunohistochemicalstaining of a panel of tissue sections from a colorectal cancer patientis conducted.

GNX-8 antigen is expressed not only on colorectal cancer tissues butalso on adjacent tissues. For example, a polyp next to a cancer regionis stained by GNX-8. However, no staining is observed on distal normaltissues. Hence, it can be concluded GNX-8 identifies transformed cellsor cells undergoing transformation before recognizable cell morphologychanges occur.

GNX-8 antigen expression is also studied on various grades of cancer.

GNX-8 antigen is expressed in each cancer stage.

GNX-8 does not bind to normal colon, rectum, stomach, small intestine,liver, esophagus, lung, prostate or breast.

Fifty-eight percent ( 44/76) of colon cancer samples are stained withGNX-8; 47% of rectum cancer samples; 57% of metastatic colon cancersamples; 53% of stomach cancer samples; 29% of esophageal cancersamples; 22% of lung cancer samples; 4% of prostate cancer samples; 17%of breast cancer samples; and 67% of pancreatic samples are stained withGNX-8. GNX-8 does not bind to small intestine, liver and kidney cancersamples.

TABLE 1 Specificity of GNX-8 on Human Normal Tissues Normal TissuesIncidence Human tissues (No. positive/No. tested) Colon  14/102Esophagus  3/4* Breast  13/56^(§) Pancreas   4/12^(#) Kidney  11/63^(#)Rectum  1/106 Small intestine 0/2 Liver 0/4 Lung 0/3 Prostate 0/6*stained on keratinization of stratified squamous epithelium ^(§)stainedon epithelial cells of duct system/lactiferous ducts ^(#)stained onepithelial cells of duct system

TABLE 2 Specificity of GNX-8 on Human Cancer Tissues Incidence (No.positive/No. Staining intensity and Human cancer tissues tested)location Colon 44/76  3 + (5), 2 + (12), 1 + (27) Rectum 50/107 3 +(13), 2 + (20), 1 + (17) Small intestine 0/10 Liver 0/12 Kidney 0/3 Colon (metastatic) 27/47  2 + (9), 1 + (18) Esophagus 4/14 2 + (1), 1 +(3) Lung 10/45  3 + (1), 2 + (5), 1 + (4) Prostate 2/45 2 + (2) Breast7/45 3 + (1), 2 + (4), 1 + (2) Pancreas 8/12 2 + (3), 1 + (5)

Example 9 Cloning and Sequencing of GNX-8

GNX-8 producing hybridoma cells are routinely cultured in IMDM(Invitrogen) containing 10% low IgG fetal bovine serum (HyClone). Toprepare RNA for cDNA synthesis, 1×10⁶ hybridoma cells are firstharvested by low speed centrifugation (1000 rpm, 5 min). Total RNA thenis isolated from the cell pellet using TRIZOL reagent (Invitrogen)according to the manufacturer's protocol. First strand cDNAs aresynthesized from the purified RNA sample using the SMART RACE cDNAAmplification Kit (BD Biosciences-Clontech). Briefly, 1 μg total RNA isincubated with 1 μg 5′-CDS and 1 μl SMART II A oligo primers at 70° C.for 2 minutes. After addition of 2 μl 5× first-strand buffer, 1 μl 20 mMDTT, 1 μl 10 mM dNTP and 1 μl of PowerScript RT are added to theRNA/primer mixture. The sample is incubated further at 42° C. for 1.5hours. The first strand cDNA synthesis reaction is terminated by adding100 μl Tricine buffer and incubated at 72° C. for 7 minutes.

The cDNA encoding the heavy chain fragment of GNX-8 is amplified by PCRusing UPM (BD SMART RACE cDNA Amplication Kit) and a primer of the 3′end of the heavy chain, CH1, SEQ ID NO:3. The PCR reaction is carriedout at 94° C. for 30 seconds, followed by 58° C. for 30 seconds, 72° C.for 3 minutes, and that cycle is repeated 26 times.

The variable region of the heavy chain cDNA is re-amplified from 1 μl ofthe above reaction product in the presence of NUP (SMART RACEamplification kit) and a primer of the middle of the heavy chain CH1,SEQ ID NO:4. The PCR reaction is performed at 94° C. for 15 seconds, 68°C. for 30 seconds and that cycle is repeated 25 times. The amplifiedproduct is purified using a PCR purification kit (GeneMark) and thenucleotide sequence is determined using a primer of the 5′ end of theheavy chain CH1, SEQ ID NO:5.

Based on the sequence information, the full length heavy chain cDNA isspecifically amplified by PCR from previously prepared first strandcDNAs with newly synthesized primers, SEQ ID NO:6, and for the end ofthe heavy chain gene, SEQ ID NO:7, with the BD Advantage 2 PCR EnzymeSystem (BD Biosciences). The PCR reaction is set at 94° C. for 40seconds, 60° C. for 30 seconds and 72° C. for 100 seconds, and thatcycle is repeated 35 times.

The amplified full length heavy chain cDNA is first double digested withEcoRI and XbaI. After gel purification, the recovered heavy chain cDNAthen is ligated into the pCIneo vector (Promega) at the same sites toobtain the expression vector pCI-GNX-8.H3. The inserted cDNA sequence isconfirmed using a primer that hybridizes upstream of the multiplecloning site, SEQ ID NO:8, and a primer downstream of the multiplecloning site, SEQ ID NO:9. GNX-8 heavy chain cDNA and the deduced aminoacid sequence are shown in Table 3 as SEQ ID NOS:14 and 15,respectively.

To identify the light chain cDNA sequences, the light chain peptide ofGNX-8 is subjected to mass spectrometry analysis and database search.According to the protein identification information, the light chain ofGNX-8 is homologous to the mouse λ chain. A primer flanking the 5′ endof mouse lambda gene constant region, SEQ ID NO:10, is synthesized. AcDNA including the variable region and a part of the constant region ofthe light chain gene is amplified from the first strand cDNAs describedpreviously by touchdown PCR. The PCR reaction is carried out first for 5cycles of 30 seconds at 94° C., 90 seconds at 72° C., followed by 5cycles of 30 seconds at 94° C., 30 seconds at 66° C., 90 seconds at 72°C., and then the cycle of 30 seconds at 94° C., 30 seconds at 63° C.,and 90 seconds at 72° C. is repeated 27 times. The amplified PCRfragments then are introduced into the yT&A vector (Yeastern Biotech)for positive clone identification.

Four clones with the expected size are selected for sequencedetermination with primer, SEQ ID NO:11.

The results indicate that all four clones have the identical cDNA with astructure homologous to the 5′ end of known light chain genes.

To reconstitute the full length light chain cDNA, a new set of primers,SEQ ID NO:12 and SEQ ID NO:13, are synthesized and the cDNA describedabove is used to prepare only the variable region of the light chaingene by PCR. The PCR reaction includes 30 cycles at 94° C. for 30seconds, 60° C. for 30 seconds and 72° C. for 1 minute.

The amplified light chain variable region cDNA with incorporated EcoRIand BsiW1 restriction enzyme sites is digested with the respectiveenzymes. After agarose gel purification, the amplified variable regioncDNA fragment is ligated into the same sites of the pCIck vector (apCIneo based expression vector with the insertion of a human κ constantregion at the XbaI and NotI sites) to give the light chain expressionvector pCIck-GNX-8.mλ. Sequencing confirmation is performed and thededuced nucleotide, as well as amino acid sequences of GNX-8 light chainare shown as SEQ ID NOS:16 and 17, respectively.

A single vector that expresses both heavy chain and light chain genes ofthe recombinant GNX-8 antibody is constructed with either the neomycingene (pCIck-GNX-8 neo) or the DHFR gene (pCIck-GNX-8 DHFR) as aselection marker. The light chain vector pCIck-GNX-8.mλ is linearizedwith BglII followed by 5′ end dephosphorylation with calf intestinephosphatase (CIP). The heavy chain vector pCI-GNX-8.H3 is cleaved withBglII and NgoMIV. The BgM-NgoMIV fragment containing the CMV promoter,full length heavy chain cDNA and SV40 polyA is recovered by gelextraction and introduced into the linearized light chain vector byblunt end ligation to form pCIck-GNX-8 neo. Subsequently, thepCIck-GNX-8 DHFR vector is generated by removing the neomycin gene withNgoMIV/ClaI cleavage from pCIck-GNX-8 neo and replacement with the DHFRgene. The DHFR minigene is cleaved from the pdhfr3.2 vector (ATCC No.37166) by HindIII/SalI digestion, and is separated and recovered by gelextraction. Both fragments are treated with Klenow to give blunt ends,then the DHFR gene is ligated into the NgoMIV/ClaI-cleaved pCIck-GNX-8neo fragment by blunt end ligation.

TABLE 3  Primers and Sequences Sequence (5′ to 3′) SEQ ID NO ELLGG 1MISRT 2 GCA TGT ACT AGT TTT GTC ACA 3 AGA TTT GGGGTG CAC GCC GCT GGT CAG GGC 4 GCC TG GGT GCC AGG GGG AAG ACC  5 GAT GGCGA ATT CAC CAT GGC TGT CTC 6 CTT CCT C GCT CTA GAT CAT TTA CCC GGA 7GAC AGG ACT CCC AGT TCA ATT ACA GC 8 TGG TTT GTC CAA ACT CAT C 9GCA TGT ACT AGT TTT GTC ACA 10 AGA TTT GGG GTT TTC CCA GTC ACG AC 11GCG AAT TCA CCA TGG CCT GGA 12 CTT CAC GCC GTA CGT AGG ACA GTG ACC 13TTG GTT C GDSVSSKSVA 18 GGGGACAGTGTCTCTAGCAA 19 GAGTGTTGCT TYYRSKWYN 20ACATACTACAGGTCCAAGT 21 GGTATAAT ARNFDY 22 GCAAGAAACTTTGACTAC 23TGAVTTNNY 24 ACTGGGGCTGTTACAACT 25 AATAACTAT ATS 26 GCTACCAGC 27ALWYNTHFV 28 GCTCTATGGTACAACACCC 29 ATTTTGTT

Length: 318 Type: DNA (heavy chain, variable region) SEQ ID NO: 14GGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAAGAGTGTTGCTTGGAACTGGATCAGGCAGTCCCCATTGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGAATATGCAGTATCTGTGAAAAGTCGAATAACCATCAATCCAGACACATCCAAGAACCAGTTCTCCCTGCACCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAAACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCC Length: 106 Type: amino acid(heavy chain, variable region) SEQ ID NO: 15GLVKPSQTLSLTCAISGDSVSSKSVAWNWIRQSPLRGLEWLGRTYYRSKWYNEYAVSVKSRITINPDTSKNQFSLHLNSVTPEDTAVYYCARNFDYWG QGTLVTVS Length: 300Type: DNA (light chain, variable region) SEQ ID NO: 16CTCACCACAGCACCTGGTGGAACAGTCATACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAATAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGATGCTACCAGCAACCGAGTTCCAGGTGTTCCTGTCAGATTCTCCGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGATGCAATGTATTTCTGTGCTCTATGGTACAACACCCATTTTGTTTTCGGCGGTGGAACCAAGGTCACT GTCCTA Length: 100Type: amino acid (light chain, variable region) SEQ ID NO: 17LTTAPGGTVILTCRSSTGAVTTNNYANWVQEKPDHLFTGLIDATSNRVPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYNTHFVFGGGTKVT VL

Once the light and heavy chains are sequenced, the nucleic acids can berecoded to optimize expression in, for example, specific human hostcells.

Example 10 Transfectomas

NS0 cells are grown to a density of 1×10⁶ cells/ml. The cells aremaintained in exponential growth phase and medium is changed the daybefore transfection. The day of transfection, 40×10⁶ cells are washed.Then, 10 μg of linearized nucleic acid containing, for example, lightchain DNA and linearized heavy chain DNA are added to the cellsuspension (the total DNA volume should be less than 50 μl) and theculture incubated on ice for 15 min. The DNA and cell mixture istransferred to a chilled cuvette (0.4 cm) and an electric pulse (750 Vand 25 μF) is applied. The cuvette is placed on ice immediately afterthe electric pulse and kept on ice for 10-15 min. The cells arecollected and plated. The cells are incubated in a 5% CO₂ incubator for12-16 days or until colonies appear. The supernatant of the cellcolonies or cells grown in suspension culture is tested by ELISA andpositive transfectomas are cloned in fresh medium. To further screen thepositive transfectomas, either titration ELISA or the Biacore assay isconducted. Expanded transfectomas are maintained in shaker flasks andantibody or derivative thereof collected from the supernatant.

Example 11 Pharmacokinetics

Rats are randomized into two groups (four rats/group). Animals receive 1or 10 mg/kg of GNX-8 as a single i.v. bolus injection via a tail vein.Blood samples are collected at 0, 5, 15 and 30 minutes, and 1, 2, 4, 6,24, 30, 72, 144, 168, 216, 240, 312, 336 and 360 hours. Serum isharvested and is stored at −20° C. until the GNX-8 concentration assay.GNX-8 concentration is determined by ELISA.

GNX-8 is radiolabeled with ¹³¹I using the IODO-Gen method.

Nude mice are used for in vivo biodistribution and imaging studies. Fivemillion Colo205 cells are inoculated subcutaneously. When the tumorreaches a size of 0.5 g, the biodistribution study is performed. For thebiodistribution study, mice are injected in the tail vein with 10μCi/2.3 μg ¹³¹I-labeled GNX-8. Five mice are sacrificed at 6, 24, 48, 72and 96 hours after injection. Blood samples are taken just before themice are sacrificed. Tumors and organs (brain, skin, muscle, bone,heart, lung, pancreas, eye, adrenal, tail, spleen, kidney, liver,bladder, stomach, small intestine and large intestine) are removedimmediately and weighed. Presence of radiolabeled antibody in tumor,organ and blood are separately counted in a y counter (Packard).Standards are counted each time with the tissues and tumors. Tissueradioactivities are expressed as the percentage of injected dose pergram of organ (% ID/g).

Imaging studies are performed on a microSPECT single photon emissioncomputerized tomography X-SPECT animal imaging system (Gamma Medica Inc.USA) and microCT X-ray computerized tomography. Nude mice bearingColo205 tumors are i.v.-injected with 200 mCi/5.5 mg/100 ml ¹³¹I-labeledGNX-8 via tail vein. Images are acquired at 1, 6, 24, 48, 72 and 96hours. Pharmacokinetic (PK) studies are conducted for GNX-8 in rats,SCID mice and nude mice following a single i.v. administration. Serumconcentration of GNX-8 is analyzed by ELISA.

A two-compartment model provides a good fit to the data and generatesthe PK parameters summarized in Tables 4 and 5. A dose-related increasein Cmax is observed following a single i.v. administration of 1.0 and 10mg kg GNX-8 in rats. GNX-8 is cleared from the serum in a terminalhalf-life of 3.81 and 4.98 days at a dose of 1 and 10 mg/kg,respectively.

The pharmacokinetic parameters of GNX-8 after administration in nudemice and SCID mice with or without Colo205 tumor-bearing mice presentedwith a T_(1/2) that is about one day in both species with tumor. Fornon-tumor-bearing animals, T_(1/2) values are 58.09 hr in SCID mice and98.31 hr in nude mice, respectively. From the pharmacokineticparameters, the T_(1/2) is much longer in the non-tumor-bearing micethan in tumor-bearing mice.

TABLE 4 Pharmacokinetic parameters in rats Parameter 1 mg/kg i.v. 10mg/kg i.v. T_(max) (minutes) 5 5 C_(max)(ug/ml) 9.49 102.08 T_(1/2)terminal (day) 3.81 4.98

TABLE 5 Pharmacokinetic parameters in SCID mice and Nude mice (i.v.administration of 5 mg/kg GNX-8) SCID mice Nude mice Tumor-bearing + − +− C_(max) (ug/ml) 76.45 72.52 76.5 84.4 T_(max) (min) 5 5 5 30 T_(1/2)(elimination) (hr) 22.99 58.09 26.73 98.31

Biodistribution studies are done in nude mice bearing Colo205 xenograftsto assess the in vivo tumor targeting activity and specificity of GNX-8.

The highest level of ¹³¹I-GNX-8 radioactivity is detected in plasma atall time points, 6 h, 24 h, 48 h, 72 h and 96 h. At 6 h, about 60% ofthe injected does per gram (% ID/g) is detected in plasma. At 48, 72 and96 hours, the % ID/g for plasma was about 20%. The plasma level issignificantly higher than in other organs, brain, skin, muscle, bone,heart, lung, pancreas, eye, adrenal gland, tail, spleen, kidney, liver,bladder, stomach, small intestine and large intestine, where the % ID/gat all time points in all organs does not exceed 5% ID/g. Radioactivitydecreases over time in plasma and in the other organs, except the tumor.Plasma radioactivity decreases by about 70% between 6 and 96 hours.

Radioactivity of the tumor is initially higher than in normal organs.The highest tumor uptake is observed at 48 hours after ¹³¹I-GNX-8injection, and maintains a steady state while the radioactivity of otherorgans decreases. Therefore, the tumor/organ ratios increase in otherorgans. Rapidly decreasing radioactivity is observed in plasma, heart,lung, adrenal gland, tail, spleen, kidney and liver between 6 and 24hours. On the other hand, tumor/plasma ratios increase about 4 timesfrom 6 to 96 hours after injection. No accumulation of ¹³¹I-GNX-8 inkidney is observed.

The in vivo tumor targeting activity in Colo205 tumor bearing nude miceis studied by imaging analysis. A time course experiment is done tomonitor the distribution of ¹³¹I-GNX-8. The result also indicates thatthe majority of ¹³¹I-GNX-8 is located in blood, and tumor targeting isclearly visible from 24 to 72 hours after injection.

The in vivo data indicate that most GNX-8 is retained in blood afterinjection. There is no significant non-specific binding in variousnormal organs. Moreover, GNX-8 targets the Colo205 tumor rapidly afteri.v. injection and maintains labeling at a steady state level over 96hours.

Example 12 Toxicity

Single dose toxicity is performed in male and female BALB/c AnN Crl BRmice (6 mice/group) at 8-9 weeks of age. Mice are i.v. injected withGNX-8 at a dose of 150 mg/kg or with vehicle alone (PBS). Body weightfor all mice is measured on study days 1, 8 and 16 prior to sacrifice.Mice are observed daily for signs of morbidity or mortality.

Repeat dose toxicity is carried out in male Sprague-Dawley Crl CD (SD)rats (6 rats/group) at 8 weeks of age. Each group is administeredvehicle (PBS) or 3, 15 or 75 mg/kg/dose of GNX-8 twice weekly for 4weeks. All animals are checked daily for mortality and any finding isrecorded individually. Rats are weighed weekly during the pretreatmentand treatment periods, and a final overnight fasting body weight isobtained at terminal sacrifice. Blood samples are collected at terminalsacrifice and evaluated for hematology and clinical chemistryparameters. The significance of differences in body weight and alltested parameters is determined by Student's t test.

To determine the cross reactivity of GNX-8 on normal human tissues, avery high dose (150 μg/ml) of biotinylated GNX-8 is used. A tissue arraywith 72 human tissues (24 types of normal organs taken from 3 normalhuman individuals) (US Biomax FDA 80′-1) are stained with GNX-8. Basedon the results from the C_(max) of the pharmacokinetic study, GNX-8 isused at 150 μg/ml to ensure that GNX-8 at C_(max) would have no seriouscross reactivity with normal human tissues. That concentration is muchhigher than regularly used in immunohistochemical studies.

Weak to moderate staining of GNX-8 is observed on several human tissuesof epithelial origin, including mucosal epithelium of thegastrointestinal tract, epithelium cells of lactiferous ducts andkeratinized cells of stratified squamous epithelium. According to theprevious findings of Finstad et al. (Clin. Cancer Res., 3:1433-1442,1997), antibodies introduced into circulating blood show specificlocalization to carcinoma cells and did not accumulate in theantigen-positive, adjacent normal epithelial cells. Also, antibodies donot traverse the basement membrane. Therefore, the staining inepithelial cells of ducts in normal tissue by GNX-8 is not considered adetriment.

Two additional studies are conducted for examining the cross reactivityof GNX-8 on normal human blood cells. All tested blood cells from thefour various blood type (ABO) donors show negative response with GNX-8.The results support that GNX-8 does not bind to blood cells. Therefore,GNX-8 administered to the circulatory system should not cause damage toblood cells.

To address the safety of GNX-8 in vivo, two studies are conducted todetermine the acute and subacute toxicity effects.

The single dose toxicity of GNX-8 is tested in BALB/c mice at a dose of150 mg/kg. Mice are observed once daily and no deaths are found beforescheduled sacrifice. All mice gain weight over the duration of the studyand there are no significant differences in mean body weight gainbetween the GNX-8 treatment group and the control group.

The repeat dose toxicity of GNX-8 is performed in Sprague Dawley Crl CDrats. Six animals of 8 weeks age are allocated to the groups. One groupis administered vehicle (PBS) twice weekly for four weeks. Groups 2, 3and 4 receive GNX-8 in PBS at 3, 15 and 75 mg/kg/dose, respectively,twice weekly for four weeks. All animals are examined daily formortality and all findings are recorded. Rats are weighed weekly duringthe pretreatment and treatment periods and a final overnight fastingbody weight is obtained at terminal sacrifice. Blood samples arecollected at sacrifice and evaluated for hematology and clinicalchemistry parameters. Significance of differences in body weight and allother measured parameters are analyzed by Student's t test.

There are no clinical signs of toxicity related to GNX-8 treatment.Analysis of final body weight gain indicates no difference betweentreatment and control groups. Blood samples of the control group and thehigh dose group are taken prior to euthanasia following an overnightfast, and are analyzed for hematology and clinical chemistry profiles.

There are no statistically significant differences between the high doseand the control groups for hemoglobin amount, hematocrit, RBC number,mean corpuscular volume, mean corpuscular hemoglobin and meancorpuscular hemoglobin concentration. The results indicate no hematologytoxicity is induced by repeated high-dose GNX-8 administration.

For clinical chemistry analyses, mean values for the parameters for thecontrol and the high-dose groups are indicated in Table 6. Astatistically significant increase in total protein is noted in the highdose group that might result from the repeated injections of high doseantibody. Additionally, a slight increase in albumin also is observed.However, the value is still within the range of normal limits for rats.The clinical chemistry data show no notable injury to metabolism andexcretion function after repeated injection of high dose GNX-8.

Both hematology and clinical chemistry analyses verify the safety ofrepeated high dose GNX-8 administration over a four-week duration.

TABLE 6 Clinical Chemistry Analyses Control High dose Items Mean SD meanSD albumin (g/dl) 3.3 0.4 3.9 0.2 ALT(GPT) (U/L) 118.6 50.0 67.6 6.0AST(GOT) (U/L) 372.0 203.8 323.3 118.2 total bilirubin (mg/dl) 0.3 0.10.6 0.2 BUN (mg/dl) 12.3 5.8 11.2 1.5 Ca (mg/dl) 11.0 0.2 11.2 0.2 Cl⁻(mmol/L) 108.0 2.1 106.0 2.2 cholesterol (mg/dl) 96.6 9.2 86.4 18.7creatinine (mg/dl) 1.2 0.7 1.1 0.5 K⁺ (mmol/L) 4.9 0.6 4.9 0.5 Na⁺(mmol/L) 144.6 2.7 148.2 4.2 phosphorous (mg/dl) 8.4 0.6 7.4 0.9 Totalprotein (g/dl) 6.3 0.3 7.1 0.2

Example 13 Scale-Up

To obtain a stable cell line for large scale production of GNX-8, NS0and CHO cell lines expressing recombinant GNX-8 (rGNX-8) are obtained.Briefly, cDNAs encoding both the heavy and light chains of GNX-8 arecloned from the original hybridoma. The isolated antibody genes then arereassembled in expression vectors. The molecular weight of rGNX-8purified from media conditioned by transfected NS0 cells is confirmed bySDS-PAGE. The specificity, binding activity and efficacy of rGNX-8 andthe original GNX-8 hybridoma are compared by HPTLC, immunostaining, flowcytometry and CDC assay.

There are no differences between the original and recombinant GNX-8antibodies.

The N-glycosylation profiles of rGNX-8 and GNX-8 then are analyzed byMALDI-MS.

The data illustrate a highly similar N-linked sugar pattern between thetwo antibodies. Almost all N-glycans of the two antibodies contain thecore fucosylation structure, but no terminal sialic acids.

Dihydrofolate reductase deficient Chinese hamster ovary (CHO/dhfr⁻)cells (ATCC CRL-9096) are maintained in IMDM containing 5% FBS andsupplemented with 100 nM hypoxanthine and 16 μM thymidine. To preparerecombinant GNX-8 production cell lines, expression vector pCIck-GNX-8DHFR is linearized by BamHI and the concentration of recovered DNA insolution is determined by OD₂₆₀ absorbance. Approximately 1.2×10⁶CHO/dhfr⁻ cells are transfected with 10 μg linearized DNA and 30 μl ofFugene6 transfection reagent (Roche) according to the manufacturer'sinstructions. After 48 hours, the culture medium is replaced with 5%dialyzed fetal bovine serum containing IMDM for transfectant selection.The selection is continued for approximately two weeks until stablecolonies are obtained. Multiple colonies are picked and cultured underthe same selective medium in 48-well plates. Individual CHO clones arescreened for rGNX-8 expression by antigen-specific ELISA usingHRP-labeled anti-human IgG(Fc) antibodies as the second antibody.

A CHO clone that expressed high levels of antibody is selected forsubsequent gene amplification with methotrexate (MTX). Geneamplification in 10 nM of MTX results in more than a 30-fold increase ofrGNX-8 secretion. The stable CHO clone is named CHO-rGNX-8.5M10.

CHO-rGNX-8.5M10 cells are later adapted to serum-free culture in shakingflasks and achieve a maximum yield of approximately 120 μg/ml of GNX-8over a 14-day culture. The culture supernatant is collected and purifiedby Protein A chromatography. The rGNX-8 antibody purified from the CHOculture supernatant by Protein A chromatography reveals the expectedlight chain and heavy chain peptide bands on SDS-PAGE.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A human monoclonal antibody or antigen binding portion thereof thatspecifically binds an epitope comprising an extended Type I chaincomprising Le^(b), wherein said epitope is expressed on a cancer cell,wherein said antibody or antigen binding portion thereof does not bindto human erythrocytes.
 2. The antibody of claim 1, which does not bindto Le^(x).
 3. The antibody of claim 1, which does not bind to Le^(y). 4.The antibody of claim 1, which does not bind to Le^(y)-Le^(x).
 5. Theantibody of claim 1, which lyses about 50% of Colo205 cells in an ADCCassay at an E/T ratio of about 20/1 at an antibody concentration ofabout 5 μg/ml.
 6. The antibody of claim 1, wherein said cancer cellexpresses Le^(b)-Le^(a).
 7. The antibody of claim 1, wherein said cancercell is an epithelial cell.
 8. The antibody of claim 7, wherein saidepithelial cell comprises colon, rectum, esophagus, lung, prostate,breast or pancreas.
 9. The antibody of claim 1, wherein said portionthereof is an scF_(v).
 10. The antibody of claim 1, comprising a κchain.
 11. The antibody of claim 1, comprising a γ chain.
 12. Theantibody of claim 1, comprising a CDR region obtained from GNX-8.
 13. Acomposition comprising the antibody of claim 1 and a pharmacologicallyactive agent.
 14. An article of manufacture comprising the antibody ofclaim 1 and a detectable moiety.
 15. A composition comprising theantibody of claim 1 and a pharmaceutically acceptable carrier, excipientor diluent.